EXPERIMENT 1 EXPERIMENT 2 BACILLI (rods)

MAGNIFICATION BACTERIAL MORPHOLOGY AND TYPES 1. Streptobacilli

• Eyepiece Magnification: 10x • 3 basic morphology of Bacteria: - Bacillus subtilis
• Low Power objective: 10x Coccus; Bacillus; Spiral - chains
• High Power objective: 40x • Bacteria multiply through binary fission, an organism duplicates - stained with Gram
• Oil Immersion objective: 100x its genetic material, or deoxyribonucleic acid (DNA), and then stain
• Eyepiece x Objective = size of object in focus divides into two parts (cytokinesis), with each new organism a. size: 4–10
• Keep both eyes open when looking through the microscope to receiving one copy of DNA.\ micrometers
reduce eyestrain. • Micrometers (μm.) is the measurement used for bacteria (μm) long and 0.25–1.0 μm in diameter,
b. shape: rod shaped
COCCI (spheres) c. color: purple
1. Staphylococci d. arrangement: small clumps
- S. aureus
- clusters or grape-like 2. Diplobacilli
masses - Mycobacterium tuberculosis
- stained with Gram stain - stained with Acid fast stain
a. size: 0.5 – 1.5 µm - pairs
b. shape: spherical a) Snapping – V shaped
c. color: purple b) Slipping- side by side Snapping
Low Powered Objective (LPO) total magnification is 10x10 = 100x d. arrangement: grape- a. size 2-4 µm length and 0.2-
like 0.5 µm width., Slipping
b. shape: rod shaped
2. Streptococci c. color: red
- S. pyogenes d. arrangement: heaps of small
- chains rods
- stained with Gram stain
a. size: 0.6-1.0 μm 3. Coccobacilli
b. shape: round-to- - Escherichia coli
ovoid coccus - short, thick, oval
c. color: purple shaped bacilli
d. arrangement: in - stained with Gram
High Powered Objective (HPO) total magnification is 10x40 = 400x chains stain
o negative
3. Diplococci because its
- Streptococcus cell wall is
pneumoniae composed
- divide to form pairs of a thin peptidoglycan layer and an outer membrane
- stained with Gram stain a. size: 1.0-2.0 µm long, with radius about 0.5 µm.
a. size: 0.5 and 1.25 μm b. shape: rod shaped
b. shape: round-to- c. color: pink
ovoid coccus d. arrangement: singly or in pairs.
c. color: purple
d. arrangement: in pairs
Oil Immersion Objective (OIO) total magnification is 10x100 = 1000x
 4. Vibrio GRAM STAINING [Escherichia coli and Staphylococcus epidermidis]
- V. cholerae 1. Flood primary stain: crystal violet for 1 minute
- comma shaped rods * All cells (Gram positive and gram negative) stains PURPLE
- Gram stain 2. Wash off after 1 minute
a. size: 1-3 µm in length 3. Flood mordant: gram’s iodine on slides for a minute
by 0.5-0.8 µm in * All cells remain purple, although a complex is formed for
diameter gram positive cells enhancing the bond between the cell
b. shape: comma wall and the primary stain. This complex is called CVI
c. color: pink complex [crystal violet iodine complex]
d. arrangement: - ( a flagellum at one cell pole as well as pili.) 4. Wash off after 1 minute
5. Decolorize slides with acetone alcohol for a few second.
SPIRAL * Gram positive cells remain purple due to CVI complex
1. Spirillum * Gram negative becomes colorless
- Campylobacter jejuni 6. Flood counterstain: safranin for 15 seconds
- appear as S-shape, bird * gram positive cells remain purple
in flight * Gram negative cells take up a pink color
- Gram stain 7. Wash off slides
a. size: 4 μm in length 8. Air dry slides before placing under microscope
by 0.2 - 0.5 μm in width
b. shape: bird in flight ACID FAST STAINING [Bacillus subtilis and Mycobacterium
c. color: pink tuberculosis]
d. arrangement: - ( a single flagellum at one or both poles) 1. Flood primary stain: carbol fuchsin for 5 mins

EXPERIMENT 3 * All cells (acid fast - positive and -negative) stains red
STAINING * Prolonged exposure allows primary stain to enter cell
1. Alcohol lamp wall
2. Flame wireloop thoroughly until red hot 2. Wash slides thoroughly
3. Get bacterial stock culture suspension 3. Decolorize slides with acid alcohol for a minute
4. Heat the mouth of tube suspension * Acid fast positive cell wall contains long chain mycolic
5. Dip the wireloop inside the tube suspension acids, rendering cells resistant to decolorization. S. epidermidis
6. Heat the mouth of the tube suspension again * Acid fast positive remains red. – very hardy microorganism, consisting of nonmotile, Gram-positive cocci,
7. Inoculate slide * Acid fast negative becomes colorless. arranged in grape-like clusters; forms white, raised, cohesive colonies
8. Heat wireloop again 4. Wash slides thoroughly about 1–2 mm in diameter after overnight incubation, and is not hemolytic
9. Repeat process for other smears 5. Flood counterstain: methylene blue on slides for 45 seconds on blood agar.
10. Heat fix all smears
* Acid fast positive remains red M. tuberculosis
11. Bring to staining area
* Acid fast negative stain blue – fairly large nonmotile rod-shaped bacterium distantly related to the
6. Wash slides thoroughly Actinomycetes. Many non-pathogenic mycobacteria are components of
For M. tuberculosis
7. Air dry slides before placing them on microscope. the normal flora of humans, found most often in dry and oily locales. The
1. Put materials inside biosafety cabinet
rods are 2-4 micrometers in length and 0.2-0.5 um in width
2. Handle sputum with caution
E. coli B. subtilis
3. Do the same process of smearing with high precaution
– coli is gram-negative rod-shaped bacteria. It is 1-3 x 0.4-0.7 µm in size and – rod-shaped, Gram-positive bacteria that show up on both positive and
0.6 to 0.7 µm in volume. It is arranged singly or in pairs. It is motile due to negative Gram stain techniques. A bacterial rod is a symmetrical cylinder
peritrichous flagella with rounded ends. A circular chromosome is typical of bacteria,
mitochondria, and plant chloroplasts
GENERAL RULES FOR GRAM STAINING REACTIONS
COCCI
• ALL are Gram positive (+)
- always have coccus in their name: Enterococcus,
Peptostreptococcus, Staphylococcus and Streptococcus.
• EXCEPT these Gram negative (-) cocci:
- Neisseria spp., Branhamella spp., Moraxella
spp., and Veillonella spp.

BACILLI
• ALL are Gram negative (-) except for:
- Mycobacteria (other than M. tuberculosis → unstainable
by Gram stain)
- Bacillus
- Actinomyces and Streptomyces
- Nocardia
- Clostridia
- Corynebacterium
- Listeria
- Lactobacillus
- Erysipelothrix

SPIRAL
• SPIRILLUM
- ALL are Gram negative (-)
• SPIROCHETE
- Difficult to gram stain due to thin cell walls → unstainable by
Gram stain

*mga odor sa microorganisms gi pangutana ni Dr. Honorio nako – jas

Organism Odor
Candida spp. Yeast
Haemophilus spp. Wet fur
Nocardia spp. Musty basement
Staphylococcus spp. Dirty sneakers
Clostridium difficile Putrid/fecal smell
Escherichia coli Floral
Corynebacterium spp. Fruity
Pseudomonas aeruginosa Grape-like
EXPERIMENT 4 COMPONENTS OF CULTURE MEDIA
DISTRIBUTION OF MICROORGANISMS IN THE ENVIRONMENT RESULTS • Food of bacteria - carbohydrates, peptones, proteins, vitamins, etc.
CULTURES GRAM STAIN RESULTS • Indicators
OBJECTIVES • Inhibitory substances for certain bacteria - dyes, antibiotics
SKIN.
1. To demonstrate the widespread distribution of microbes in the • Control pH
environment, on and in our bodies
2. To describe the characteristics of difference colonies growing on the CLASSIFICATION OF CULTURE MEDIA according to:
nutrient agar 1. Physical state
3. To perform simple streak method in the study of bacteria • Solid - substances mixed in a solidified agar (2-3%), or gelatin (1-
1.5%), or albumin matrix
MATERIALS • Semi-solid - 0.5-1% agar added to the media
1. Nutrient agar in petri dish • Liquid - do not contain solidifying substances
2. Sterile cotton swabs 2. Composition
3. Sterile plain saline solution COIN. • Synthetic - exact chemical composition of ingredients is known
• Non-synthetic - precise composition of some or all of the nutritive
PROCEDURE
elements used not definitely known
1. Moisten a sterile swab in sterile NSS and swab the ff:
• Living tissue - living cells are present
a. Skin
3. Use
b. Nostrils
• Simple
c. Ears
o not enriched; ordinary organisms easily cultivated
d. Hair
• Enriched
e. Coin
o infusion-based media; enriched with additives
f. Ballpen
• Differential
g. Cover of book
o contain dyes, sugars, and indicators to elicit a
h. Dirty lab gown
characteristic a biochemical response used to
i. Top of lab table
differentiate groups of organisms
j. Technician’s counter
• Highly Selective
2. Open petri dish but never completely remove its top lid. Lift the lid
o promote growth of a specific organism
just high enough to make complete strokes.
• Transport
3. Inoculate the swab on the agar by simple streaking →
TECHNICIAN’S COUNTER. o prolong the survival of microorganisms when a significant
4. Incubate the plate upside down at 37 C for 24hrs
delay occurs between collection and culturing
- agar containing portion up
• Enrichment
5. After incubation, examine different colonies. Note size, shape, margin,
***GIKAN SA NOTES NI DR. CEBRECUS o designed for stool culture where normal flora are
elevation, color, opacity, consistency, texture, and odor*.
suppressed favoring growth of enteropathogens
6. In the next laboratory period, perform Gram staining of the different
PURPOSES OF CULTIVATION
types of colony. PREPARATION
• To grow and isolate all bacteria present in an infection
• To determine which of the bacteria that grew are most likely • Available in dehydrated (powder or granule) or tablet form
causing the infection and which are likely contaminants or • Agar containing media heated to dissolve agar using boiling water
colonizers bath or steam
• To obtain sufficient growth of clinically relevant bacteria to allow • Containers
identification and characterization - Petri dish (plate) - solid; provides a large surface area
- Test tubes - solid, semi-solid, liquid; cotton plug/screw-capped
PRINCIPLE
• Cultivation is a process of growing microorganisms by taking STERILIZATION
bacteria from the infection site by some means of specimen • Should be sterile prior to use
SKIN. TECHNICIAN’S COUNTER. COIN.
collection and growing them in the artificial environment of the • Most common method: Autoclave
laboratory • Inspissation - used for media containing serum or certain proteins
• Required nutrients are supplied in a culture medium • Filtration - used for carbohydrate solutions and other liquids that
• Organisms that grow and multiply in or on a culture medium are may be denatured by heat
referred to as culture
STORAGE 5. Changes in Agar Media PROCEDURE (Escherichia coli – Bacillus subtilis)
• Refrigeration - Hemolytic pattern on Blood Agar (BA) 1. Nutrient broths should be properly labelled with the specific bacteria
• To prevent deterioration and dehydration - Changes in the color of the pH indicator and its respective physical agent.
• Allowed to come to room temperature prior to use - Pitting of agar surface 2. After precuring needed materials, make a smear for each bacterial
6. Consistency and Texture suspension.
PROCESS OF BACTERIAL CULTIVATION - Dry and Friable - can be pushed around the surface of the 3. After smearing, airdry to preserve their bacterial morphologies
Inoculation medium followed by heat fixation to prevent wash off during staining.
• Implantation or introduction of specimen into the culture medium - Viscous - clings to wire when touched; stringing away 4. Transfer an impregnated nutrient filter paper disk into a nutrient broth
• Instruments: upon withdrawal labelled control. This will serve as our control and we’ll not subject this
o sterile cotton swab - Smooth - appears homogenous to any physical agent which may inhibit the bacteria’s growth or may
o inoculating wire loop/needle - Mucoid - water-like, confluent; (+) slime layer or capsule destroy them all together.
• Inoculum: organism or specimen introduced into culture medium in organism 5. Transfer the same impregnated filter paper disk to the nutrient broth
• Specimen to be inoculated into: - Rough - surface may be striated or granular labelled boiling (to be placed in a 100C water bath for 30 minutes), and
o Liquid (broth) - suspend and mix 7. Odor cold (to be placed in the refrigerator overnight).
o Solid (agar) - streak, stab, stab and streak 6. Take the impregnated filter paper disks which was already placed in the
METHODS OF OBTAINING PURE CULTURE hot air sterilizer for 160C for 1 hour and transfer this to the Nutrient
Incubation Streak Plate broth labelled hot air.
• Provide the proper temperature and ventilation • Most practical and most useful method of obtaining 7. Take the filter paper disk which was already autoclaved for 120C for 15
• Usually for a period of 18-48 hours at 37ºC discrete/isolated colonies and pure culture minutes under 15 lbs. of pressure and transfer this to the Nutrient
• If specimen contains only one organism, a pure culture will be broth labeled autoclave.
INSPECTION OF CULTURES
obtained after incubation of primary plates 8. After which, all are incubated overnight at 37C for 24 hours.
• Indications of Growth in a Broth
• If specimen has a mixed population, a pure culture will only be
- Turbidity
obtained upon subculture of a single, isolated colony from the RESULTS Turbid: there is bacterial growth (+)
- Change in color
primary plate (single colony subculture [SCSC]) Clear: no bacterial growth/killed by physical agent (-)
- Gas bubbles
• Indications of Growth on Agar METHOD AGENT TIME
- Bacteria that have multiplied will cling together to form a Boiling 100 C 30 Protein Has more rapid
visible mass known as colony water minutes coagulat- action and more
ion penetrating power
CRITERIA TO CHARACTERIZE BACTERIAL COLONIES compared to dry
1. Size heat
- Usually expressed in mm Dry heat 160 hot 1 hour Protein other methods
- Relative terms: pinpoint, small, medium, large (hot air) air oxidation which use the same
2. Pigmentation sterilizer principle include
3. Shape Pour Plate or Spread Plate
direct flaming of
- Form, Elevation, Margin • Used as a means of determining the approximate number of
hoops and
viable organisms in a liquid like water or milk
incineration of
• Consists of the preparation of a series of dilutions of the specimen
biological wastes
• Result is expressed as the number of colony forming units (CFU) per
Autoclave 120 C 15 Protein the most powerful
mL [CFU/mL] of the substance tested
(moist under 15 minutes coagulat- heat sterilizing
EXPERIMENT 5 heat lbs. of ion agent that even
GROWTH INHIBITION & DESTRUCTION OF BACTERIA BY PHYSICAL AGENTS under pressure vegetative spores
pressure) are destroyed
PREPARATION Cold Refriger- overnight Bacteriostatic effect
1. Suspension of Bacillus subtilis and Escherichia coli ated i.e. capable of inhibiting the
2. Filter paper disk impregnated with both organisms growth or reproduction of
3. Filter paper disk impregnated with both organisms placed in hot air bacteria but is not capable of
sterilizers; killing them
4. And another set which was autoclave
4. Surface Appearance 5. Nutrient broths
- Glistening, Dull, Translucent, Opaque
Escherichia coli – heat sensitive Bacillus subtilis Physical Agent Control Observation Interpretation
Bacillus subtilis
Moist heat (boiling) Turbid Turbid Bacteria not killed
Autoclave Clear Clear Bacteria killed
Dry heat Clear Clear Bacteria killed
Cold (refrigerated) Turbid Turbid Bacteria not killed
Clear; ND Escherichia coli
Clear; ND AUTOCLAVE Moist heat (boiling) Clear Clear Bacteria killed
Autoclave Clear Clear Bacteria killed
AUTOCLAVE
Dry heat Clear Clear Bacteria killed
Cold (refrigerated) Turbid Turbid Bacteria not killed

• Killing effect of heat

o above the growth range temperature of a microbe
enzymes and other proteins as well as nucleic acids are
Clear; ND denatured
o heat drives off water causing death of water dependent
Clear; ND BOILING organisms
BOILING • Killing rate of heat
o function of time and temperature
• Thermal death time
o time necessary for killing the population at a given
temperature
Turbid; Gram (+) • Thermal death point
Turbid; Gram (-) o minimal temp at which it dies in a given time
streptococci
coccobacilli MOIST HEAT
COLD COLD - kills by coagulation of proteins (enzyme bonds and structural
proteins) which is caused by breakage of H
- bonds between peptide occurs quickly in the presence of water
because water molecules conduct heat better
- much more rapid than dry heat
- can be used at lower temperature and shorter exposure time
- causes protein denaturation which results in destruction of the
Clear; ND tertiary structure of protein and become a
Clear; ND - two-dimensional structure, they coagulate and become
HOT AIR
nonfunctional.
HOT AIR - protein coagulation requires less energy than oxidation, less heat is
needed
- when using boiling water, a minimum exposure of 30 minutes must
be followed
BOILING
- a form of moist heat
- 15-30 minutes at 100 C -adequate to kill vegetative forms but not
spores
Turbid; Gram (+) - addition 2% sodium carbonate increases killing effect of boiling
Turbid; Gram (-) streptococci 62 C/CLASSIC PASTEURIZATION
- form of moist heat
coccobacilli GRAM (+) - method to destroy disease-producing organisms without damaging
GRAM (-) other important
- compounds in a medium such as vitamins or nutrients in milk.
 - 62 C for 30 minutes followed by rapid cooling - Escherichia coli suspension
o to prevent damage to other necessary proteins Procedure
AUTOCLAVE 1. Prepare smear and perform gram staining.
- moist heat in the form of pressurized steam - Gram staining (turbid control)
- increase in pressure = increase in temperature o to check morphology and confirm presence of bacteria
- water molecules in steam become more energized & their o to confirm if morphology is consistent with chosen microbe as it
penetration increases. may have contamination resulting to bacterial growth
- steam under pressure 2. Transfer 1mL of E. coli suspension to a tube of 10mL disinfectant. Time
- 121 C 15lbs steam pressure/in.(psi) or 126 C 20lbs steam pressure for 5 minutes.
for 15-20minutes (media & instrument 3. Inoculate a loopful of E. coli suspension to another lactose broth. Label
- Flash autoclave - unsaturated steam at 134 C for 3 minutes (used as “control”.
in operating rooms) 4. After 5 minutes, transfer an inoculum from the inoculated disinfectant
- 132 C for 30-60 min. (infectious medical waste) to the Lactose broth tube labelled “(disinfectant) 5’”.
- *QC: Bacillus stearothermophilus in vials or spore-strip-set tests 5. After 10 minutes, transfer an inoculum from the inoculated disinfectant
DRY HEAT to the Lactose broth tube labelled “(disinfectant) 10’”.
- causes protein oxidation 6. After 15 minutes, transfer an inoculum from the inoculated disinfectant
- lethal effects will also include toxic effects of increased level of to the Lactose broth tube labelled “(disinfectant) 15’”.
electrolytes 7. After 30 minutes, transfer an inoculum from the inoculated disinfectant
- req. higher temp and longer periods to the Lactose broth tube labelled “(disinfectant) 30’”.
- uses radiating heat 8. Repeat for all disinfectants.
- does not penetrate materials easily 9. Incubate all inoculated Lactose broths at 37 C for 24 hours.
- longer periods of exposure at higher temperatures *Perform gram staining on all turbid tubes with or without color change.
- a period of two hours is required to destroy bacterial spores
- does not corrode sharp instruments & erode ground glass surfaces Results
of non-disposable syringes
- changes microbial proteins by oxidation reactions and creates and
arid internal environment
- which burns the microorganism slowly
HOT AIR
- use of ovens
- carbonization of organic material and spore destruction
- 160 C - 180 C for 1.5-3 hours
- sterilization of glassware for lab use
- oil, petroleum and powder sterilization(media preparation)
LABORATORY 6
ACTIONS OF DISINFECTANTS ON MICROORGANISMS
Materials:
Escherichia coli Gram (-) coccobacilli .
- Alcohol lamp
- Pipette After incubation, observe for bacterial growth.
- Wire loop - Growth can be distinguished by the presence of pink color in the test Indications of bacterial growth:
- Disinfectants: tube. - Pink color
o 5% Phenol - The Lactose broth contains Andrade’s Indicator - Turbidity
o 10% Betadine o Presence of acid (Lactic acid) = pink color - Gas production (H and CO 2 )
o Merthiolate o Gas production is indicated by the presence of bubbles and
o %% Lysol displacement of the medium in the small, inverted tube Mechanisms of action: (chemical agents)
o 3% Hydrogen Peroxide o Test tube used: Durham tube - Protein coagulation
o 1% Na hypochlorite Used to detect production of gas by microbes - Alteration of protein structure
o 70% Ethyl alcohol - Acid – pink color o Protein oxidation
o Zephiran Cl- 1:1000 - Alkaline – yellow color - Cell membrane damage
- Lactose broth
 READING Antiseptic vs Disinfectant Procedure:

SUSPEN-SION
GRAM STAIN
CONTROL 5’ 10’ 15’ 30’
DISINFECT-
ANTS Antiseptic 1. Get from the counter an antibiotic sensitivity test result of a known
o destroy or inhibit bacterial growth organism.

Gram

Gram

Gram

Gram
Gram
Obs

Obs

Obs

Obs

Obs
does not necessarily kill microorganisms 2. Examine the plate and measure the diameter to the nearest
o lower concentration millimeter the zone of complete bacterial inhibition around the
o On skin antibiotic disc
Disinfectant
E. Coli Gram (-)

Bacteria killed
%5 Phenol

coccobacilli
coccobacilli

after 5 mins
o Death to disease-producing microbes; kills
Gram (-)
o higher concentration

ND

ND

ND

ND
AG

NC

NC

NC

NC
o On inanimate objects
Sterilizing agents:
- Aldehydes
E. Coli Gram (-)

*Zone of Inhibition - area around the antibiotic disk in which

coccobacilli

killed after 5
- Ethylene oxide
coccobacilli
Betadine

Gram (-)

bacterial colonies do not grow

Bacteria
10%

ND

ND

ND

ND
AG

NC

NC

NC

NC
Appearance of bacterial growth after death of bacteria: *kanang pink nya mo
Reading and Interpretation
yellow nya mo pink nasad balik
- The zone diameter of the zone of inhibition for individual
- May indicate contamination of the bacteria of interest
antimicrobial agents are translated into susceptible, intermediate
Remember: or resistant categories by referring to an interpretative table such
E. Coli Gram (-)
Merthiolate

coccobacilli

killed after 5
coccobacilli

1. Volatility of agent – too high can be harmful as the Kirby-Bauer chart.

Gram (-)

Bacteria
2. *Kung wala natarong ug inoculate – no bacterial growth
ND

ND

ND

ND
AG

*Each antimicrobial agent has a specific measurement for translation.

NC

NC

NC

NC
3. Higher concentrations of disinfectants Sensitive/Susceptible
(ex. for Ethyl alcohol, more than 70%) • agent may be appropriate choice for treating the infection
- Enables faster protein coagulation making penetration harder for Intermediate
disinfectants to enter the microbial cell • agent may still be effective but usually at higher
E. Coli Gram (-)

coccobacilli

killed after 5
%5 Lysol

coccobacilli

Gram (-)

4. Ethyl vs Isopropyl alcohol:

Bacteria
concentrations
ND

ND

ND

ND
AG

NC

NC

NC

NC

- Difference is based on structure (molecular weight) Resistant

- Higher molecular weigh = higher germicidal action • agent may not be an appropriate choice for treating the
*refer to notes on Chemical agents pag lecture for more info hahaha charot infection
- The end point of all reading systems: complete inhibition of
E. Coli Gram (-)

coccobacilli

growth disregarding tiny colonies which can be detected only by

3% H 2 O 2

coccobacilli

coccobacilli

coccobacilli

coccobacilli

killed after

EXPERIMENT 7
Gram (-)

Gram (-)

Gram (-)

Gram (-)

Bacteria

very close scrutiny

ND
AG

AG

AG

AG

ANTIMICROBIAL SUSCEPTIBILITY TEST: DISC AGAR DIFFUSION TEST

NC

Objectives: Instructions:
- To determine the sensitivity to antimicrobial agents of commonly 1. Organism assignments by group in order:
isolated, rapidly growing pathogens such as: I. Escherichia coli
Hypochlorit

E. Coli Gram (-)

II. Proteus vulgaris

coccobacilli

killed after 5

o Members of the family Enterobacteriaceae

coccobacilli

Gram (-)
1% Na

Bacteria

o Staphylococcus aureus III. Salmonella enteritidis

ND

ND

ND

ND
AG

NC

NC

NC

NC

o Pseudomonas aeruginosa IV. Salmonella paratyphi A

Materials: V. Shigella dysenteriae
- Broth suspension of different organisms VI. Klebsiella pneumoniae
- Mueller Hinton Infusion agar plates VII. Staphylococcus aureus
E. Coli Gram (-)

coccobacilli

killed after 5
70% Ethyl

VIII. Pseudomonas aeruginosa

coccobacilli
Alcohol

- 5 antibiotic discs namely:

Gram (-)

Bacteria

2. Draw as accurately as possible the test result of your assigned

ND

ND

ND

ND
AG

*label the disk using their abbreviations

NC

NC

NC

NC

o Ampicillin (AM) organism.

o Gentamicin (CN) 3. Label disks used and the diameter of the zone of inhibition around
o Penicillin (P) the antibiotic disc with the correct unit of measurement.
o Chloramphenicol (C)
E. Coli Gram (-)

*Supplemental Notes ni Doc Cebrecus

coccobacilli

killed after 5
Zephiran

coccobacilli

Gram (-)
1:1000

o Trimethoprim-Sulfamethoxazole (SXT)
Bacteria

Antimicrobial Susceptibility Testing

ND

ND

ND

ND
AG

NC

NC

NC

NC

- Sterile cotton swabs Procedures used to produce antimicrobial susceptibility profiles and detect
- Forceps resistant to therapeutic agents.
- Ruler or vernier caliper
Primary goal:
To determine whether the bacterial isolate is capable of expressing
resistance to the antimicrobial agents selected for treatment.
Inoculum preparation:
- Standard inoculum size
o Comparing the turbidity of the organism’s suspension
with a turbidity standard
- McFarland Barium sulfate No. 0.5
o 0.5 - provides optical density (visually) comparable to the density
of a bacterial suspension of 1.5x108 CFU/mL
o Barium sulfate (BaSO 4 ) = 1% Barium chloride (BaCl 2 ) + 1% Sulfuric
Acid (H 2 SO 4 )
o the reaction between these two chemicals results in the
production of a fine precipitate, enabling it to be visually
comparable
o Why was this used?
Gives satisfactory growth of the majority of rapidly
growing pathogens
Not antagonistic to the major microbial agents
*McFarland Standards are used to standardize the approximate number of
bacteria in a liquid suspension by comparing the turbidity of the test
suspension with that of the McFarland Standard (a reference) – for
antimicrobial susceptibility testing and culture
media performance testing
Medium of choice
- Mueller Hinton Infusion Agar (MHIA)
o Gives satisfactory growth of the majority of rapidly
growing pathogens
o Not antagonistic (does not harm) the major antimicrobial
agents to be tested
o Isotonic with blood – can be supplemented with 5%
defibrinated blood for testing for fastidious organisms
- Depth of agar can affect test accuracy
o Uniform depth: 4 mm
o Too thick = smaller zone sizes (false resistant)
o Too thin = larger zone sizes (false sensitive)
o Too wet = faster diffusion; false susceptibility
o If medium has dried up = slower diffusion; false resistance
*agar thickness and hydration impact zone of inhibition through influencing
diffusion of the antimicrobial agent.
*thick agar = lower antimicrobial concentration; thin agar = higher
antimicrobial concentration
Inoculation of Test Plate
- Use sterile nontoxic swab
- Very close streaking
- 3 different direction – turn plate each time at 60
- Final sweep to the agar rim with cotton swab
- Allow plate to stand on a flat surface for 3-5 minutes (but not more
than 15 minutes)
o Why? – to avoid having the disc diffuse or spread right away
- Purpose: to obtain an even and complete distribution of inoculum
over the entire plate (petri dish)
Application of Antibiotic Discs
15 mm
20 mm
- Within 15 minutes after inoculation
*To avoid overgrowth of bacteria
- Gently press all discs onto agar with forceps to ensure complete
contact with the agar
- Follow special arrangement:
o 15 mm for the edge
Questions:
o 20 mm apart
Why does bacteria develop resistance to antimicrobial agents?
*to prevent zones from overlapping
• Every time an organism is treated with antimicrobial agents,
Incubation sensitive bacteria are killed, but resistant germs may be left to
- Within 15 minutes after application of discs grow and multiply.
- Follow strictly the incubation time: 16-18 hours • Due to mutation of microorganism:
o May be increase beyond 18 hours (20-24 hours) to enhance 1. Repeated, overuse, or misuse of antimicrobial agents; or
detection of certain fastidious organisms inadequate dosage to fully kill microorganism
o Why 16-18 hours? - To avoid having the discs develop *Mao na istress gyud sa imo client na tiwason ang gi prescribe
resistance to the antibiotics due to prolonged exposure na regimen sa doctor kay mao ni common cause of bacterial
resistance
Reading and interpretation:
2. Microorganism may rely or require on the agent for
- Read zone of inhibition to the nearest millimeter
growth
- Interpretative categories are assigned:
o Sensitive – antimicrobial agent may be appropriate choice for Antimicrobial: destroy microorganisms (microscopic)
treating the infection Antibacterial: destroys or inhibits bacterial growth (subset of antimicrobial)
o Intermediate – antimicrobial agent may still be effective but
Penicillin: Alexander Fleming (from Penicillum)
usually at higher concentrations
o Resistant – antimicrobial agent may not be an appropriate What is the cause of an unclear zone?
choice for treating the infection - Resistance of microbe
Not susceptible (NS) – not under resistant or intermediate - Contamination
Susceptible Dose Dependent (SDD) – would depend on client’s - Special characteristic of microorganism (flagella, etc.)
regimen of antibiotics *check gyud always sa purity sa imo suspension kay basig contaminated siya
daan
Broad spectrum antibacterial agents:
- Ampicillin

GENTAMICIN

AMPICILLIN

CHLORAM-
PHENICOL
PENICILLIN

TMP-SMX
Narrow spectrum antibacterial agents:

Group
- Penicillin (gram + only) and gentamycin
ANTIBIOTIC

G
In the results, why didn’t we record it as 0 mm if there is no zone of
inhibition that was observed?
- The disc size used is 6mm so we measure only the diameter of the BACTERIA MM INT MM INT MM INT MM INT MM INT
disk even if wala kay makita na zone because we are assuming that 1 Escherichia coli 7 R 16 S 28 S 6 R 33 S
there is a zone of inhibition underneath the disk. Proteus
2 vulgaris
8 R 15 S 20 S 6 R 24 S
If the ZOI overlaps or is too big that it reaches the side of the plate, what is Salmonella
an alternative way in measuring? 3 enteritidis
25 I 17 S 24 S 24 S 39 S
- Find the radius. (radius + radius = diameter) Salmonella
4 paratyphi A
21 I 18 S 28 S 23 S 38 S
How to check if bacteria in ZOI is killed? Shigella
- Remove the discs 5 dysenteriae
9 R 14 I 26 S 18 S 33 S
o Growth = it was only inhibited Klebsiella
6 pneumoniae
6 R 17 S 6 R 6 R 35 S
o No growth = destroyed
Staphylococcus
Do not take everything at face value. 7 aureus
7 R 14 I 32 S 8 R 25 S
There may be other possible factors that may alter the preference to use a Pseudomonas
8 aeruginosa
6 R 15 S 6 R 6 R 21 S
specific antimicrobial agent. Ex. Chloramphenicol, though it was found to be
effective, has diverse side effects on the client and should not be used with
urine.

Staphylococcus = MHIA + salt

Streptococcus = MHIA + blood

What does the 15-15-15 rule mean?

Following the EUCAST, in conducting a Disk Diffusion Test, we should adhere
to the 15-15-15-minute rule to get a reproducible result. The rule would
indicate as follows:
• Use of the inoculum within 15 minutes of preparation.
• Application of the discs within 15 minutes of inoculating the plates.
• Starting incubation within 15 minutes of the application of disks.

What would happen if the incubation time exceeds the time of 16-18 hours?
It is better to have a culture with minimum of dead cells, early stationery
phase is the best. Incubating longer would result to many dead cells
contaminating your preparation. If a bacterial culture is left in the same
media for too long, the cells consume the available nutrients, excrete toxic
metabolites, and ultimately the entire population will die. Therefore,
bacterial cultures must be periodically transferred, or sub-cultured, to a new
media to keep the bacterial population growing.
EXPERIMENT 8 3. What happens if you transfuse a different blood type?
AGGLUTINATION TEST - If you transfuse blood that's incompatible with your blood type,
your immune system might start to rip the foreign blood cells
Learning Objectives: apart, triggering a cascade of reactions which potentially include
1. To explain what an antigen-antibody reaction is blood clots clogging up your veins and killing you.
2. To perform an agglutination test Usual reactions:
Materials: o Chills
1. Anti-A and Anti-B typing sera Sample 1 o Fever
2. Rh typing serum o Urticaria (hives)
3. Clean glass slide Stop transfusion immediately if symptoms occur. Some symptoms resolve
4. Lancet with little or no treatment. However, respiratory distress, high fever,
5. Cotton hypotension (low blood pressure), and red urine (hemoglobinuria) can
6. Toothpick or Stick indicate a more serious reaction.
Procedure:
1. Divide a clean glass slide into three. Label them as “A” “B” and 4. Do you think all antigen and antibody reaction produce
“Rh”. agglutination? What term do we give to antigens and antibodies
2. Obtain specimen; either venous blood in EDTA tube or fresh Sample 2 that produce agglutination?
capillary blood can be used. - (???)
3. Place a drop of blood on each of the divided portions of the glass
slides. 5. Of all the antigens and antibodies that produce agglutination, do
4. To the drop of blood labeled “A”; add a drop of anti-A typing you think all antigen and antibody rxn would produce visible
serum. agglutination in vitro?
5. To the drop of blood labeled “B”; add a drop of anti-B typing - No
serum.
6. To the drop of blood labeled “Rh”; add a drop of Rh typing serum. 6. What are the two types of blood typing? State their differences.
7. With a clean stick, mix well the drop of blood and typing serum. Sample 3 Forward Typing
8. Record the results. - for the expression of A and B antigens on red cells by
hemagglutination
Results: - Reagent: Serum (where the antibodies are found)
There is a reaction/clumping when serum and blood are mixed - Sample: RBC/blood (surface is where antigens are found
(agglutination).
Reverse Typing
- Clumping in A = Blood type A
- to detect the presence of naturally occurring anti-A and anti-B
- Clumping in B = Blood type A
antibodies
- Clumping in Rh = Rh positive Sample 4
- Reagent: A and B cells (from those with that specific blood type)
- Sample: Serum
Questions:
1. Case: Mom is Rh (-), father is Rh (+) and baby is Rh (+). There is a Rh 1.2. When should you give the injection if there is an incompatibility for
Rh? *What was used in the experiment?
incompatibility. First pregnancy was fine. Pero ngano sa iyang 2nd
- 1st pregnancy - Forward typing
pregnancy na affected which resulted to the death of the baby?
- Mother had formed antibodies (sensitized). Her antibodies
1.3. Why is Rh factor usually a concern during pregnancy? 7. An A+ patient’s result for reverse typing is, Anti-A (+), Anti-B (-), Anti-D
viewed the baby (rather ang antibodies??? Di ko sure) as foreign.
- D (+), Rh (+). In reverse typing, which among these would agglutinate?
So ang nahitabo, gi attack ang ni siya sa antibodies sa mother
- B agglutinates
causing anemia
- Hemolysis – destruction of RBC 2. What are the steps of agglutination?
- Sensitization: antibodies coat surface of RBC by binding to Reverse typing
antigen Type A+ → B agglutinates
1.1. What injection is administered to prevent mother from forming Anti-B
- Lattice formation: antibodies bind adjacent RBC lattice forming Type B+ → A agglutinates
antibodies?
visible agglutination Type AB+ → none
- RHOGAM injection
Type O+ → both agglutinates
8. If in forward typing, O+ ang patient but in reverse typing, A is negative 13.1. Until when are we able to read the results? What is our time limit? 25. Possible complications to the sample if venipuncture? What to do?
but B is positive. Why? - Pull back vacuum negative pressure. Forceful pressure may cause
- Not possible bc if sa forward kay O+ (negative sa both anti-A and - Can read until 2 minutes for forward typing. If > 2 minutes, there lysis of RBC)
anti-B), supposedly sa reverse positive ang A and B. would be drying. Slide method is prone to drying.
26. Significance of blood typing or organ transplantation
8.1. What should be done? Unsay cause? 14. Do antibodies destroy antigens? If yes, how? - Recognize organ as foreign and destroy organ
- Basig contamination daw. Retake sample. - Yes.
- When antibodies bind to antigens it forms an immune complex. 27. What other anti a & anti b body class belong to?
8.2. In a few hours, ni retake nakas sample but the results are the same B+ Carried for disposition because of extravascular hemolysis. - igM
gihapon. Eventually leading to lysis or destruction of RBC’s - Rh
- It is because there is a presence of the weak ABO subgroups. - IgG
15. Do you want agglutination to occur inside body of patient?
8.3. What is the stronger subgroup of A? (80% sa population) - No. 28. Which crosses placenta?
- A1 ang stronger. Weaker ang A2. - IgG
16. When transfusion reaction occurs, how often should you monitor
8.4. How do you remedy this? client? Supplemental notes from Dr. Honorio
- Include serum na polyclonal (Anti-A, B). If mo agglutinate gani - 1st hour – q 15 minutes ABO SYSTEM
siya ani meaning there is a presence of A2 antigens. - 2nd hour – q 30 minutes Historical perspective
Karl Landsteiner discovered the first human blood group system,
What we used na Anti-A sa forward typing is serum with A1. It 17. How many hours till transfusion happens? the ABO.
cannot detect the A2 antigen. 80% of the population has A1 - 2-4 hours usually 4 hours The 1st blood groups he described were groups A,B,&O
antigen, 20% has A2. So if you have a patient who’s A+ but the - Platelets 30-60 min In 1902, Landsteiner’s associates discovered the 4th ABO blood
antigen is A2, you must perform both forward and reverse typing group, Group AB.
para ma detect ang A2. 18. What happens when antigen binds to antibody? What part of the
antibody binds to antigen? Two unique features not found in other blood group system:
9. As a nurse, we all know that blood typing is very important especially - (???) - The usual presence of strongly reactive agglutinins in the
in blood transfusion. It is our responsibility to always check for serum of those who lack the corresponding antigen(s).
identifiers. In a case where we fail to give the correct bag to the 19. What are other alternative ways to detect blood type? - The regular presence of ABH antigens on many tissue cells &
patient. What is our nsg responsibilities/interventions? - ABO Tests ABH substances in the secretions of secretors
- Immediately stop transfusion. - Rh Tests
- Notify the physician. - Cross Matching Antigens in certain tissue
Blood group Agglutinins in serum
- Monitor patient’s vital signs/condition q 15 minutes. cells
20. Why is venous blood in EDTA used in blood typing? Is it easier to Bombay Anti-H, Anti-A, Anti-B -
10. Antigen for blood type AB. (universal recipient) obtain venous blood than capillary blood? O Anti-A, Anti-B H(O)
- Antigen A and B. - No, because only need to prick finger to obtain sample A Anti-B H, A
B Anti-A H, B
11. Antibody for blood type AB. 21. Is arterial blood different from capillary? Different results? AB none H, A, B
- No antibodies. - No. Antigen present in patient’s blood type same
If an antigen(Ag) is present on a patient’s red blood cells the corresponding
12. Universal donor? Why? 22. What other terms agglutinogen and agglutinin? antibody(Ab) will NOT be present in the patient’s serum under normal
- Type O. Does not contain any antigens. - Agglutinogen- antigen conditions.
- Agglutinin- antibody
13. When we read the results via forward typing slide method. Would we MEDICAL IMMUNOHEMATOLOGY
read it right away after mixing reagent and sample? 23. What are advantages and disadvantages of using capillary blood The blood groups are well established at the time of birth & blood
- We don’t read it right away bc there are steps before rather than venous blood? grouping can be done from cord blood or anytime thereafter.
agglutination occurs. We wait for those steps to finish and give - Easier to prick The isoagglutinins of ABO system can be detected with confidence at age 3
time for those samples and reagents to react. –6 months, the titer reaching maximum at 5-10 years old
24. How are venous samples obtained? Reverse typing is not routinely done on newborns
- Uses a syringe – venipuncture (vein)
 Glycosyltransferase
Nucleotide
Immunodominant
- The reverse (serum) grouping procedure to confirm ABO blood 5. Plasma (medium for Coagulase test)
Gene (sugar Antigen grouping is based on the presence or absence of the antibodies , 6. Sterile normal saline solution (NSS)
(transferase enzyme) sugar
donor) anti-A and anti-B ,in the serum. 7. Hydrogen Peroxide
Alpha 2 L- - If these antibodies are present in serum, agglutination should be 8. Inoculating wire loop and wire needle, alcohol lamp
H GDP-Fuc L-fucose H
fucosyltransferase demonstrated when the patient’s serum is combined with
Alpha 3 N- reagent erythrocytes expressing either A or B antigen.
UDP- N-acetyl-D- S. aureus S. epidermidis
A acetylgalactosaminyl- A - Reverse typing is to crosscheck for forward typing.
GalNac galactosamine COLONIAL CHARACTERISTICS OF BA
transferase - Because of the lack of synthesized immunoglobulins in newborn
Alpha 3 – 0 - and very young infants, this procedure is not performed on
B UDP-Gal D-galactose B
galactosyltransferase specimens from these patients.

ABO BLOOD TYPING ABO REVERSE TYPING

PRINCIPLES OF ABO FORWARD TYPING A B O Isoagglutinins
- The ABO groups represent the antigens expressed on the Blood Group
CELLS CELLS CELLS present
erythrocytes of each group. When an antibody & its + + + Anti-A, Anti-B, Anti-H Bombay
corresponding antigen are combined in vitro, clumping of the + + - Anti-A, Anti-B O Rh +
RBCs expressing the antigen results. - + - Anti-B A Rh +
- An antibody & its corresponding antigen are not normally present + - - Anti-A B Rh +
in the same blood specimen.
- - - None AB Rh + HEMOLYTIC PROPERTY ON BA
- Human erythrocytes expressing either A or B antigens agglutinate
in the presence of reagent antisera containing IgM anti-A or anti-
DIFFERENCES
B.
Forward Typing Reverse Typing
Anti-A will agglutinate all RBCs expressing A antigen.
- Cell grouping - Serum grouping
Anti-B will agglutinate all RBCs containing B antigen.
- Determination of ABO grouping is important in pre-transfusion Defined as using reagent red
Defined as using known sources
studies of patients & donors as well as specialized cases ( cells with known ABO antigens
of reagent antisera (antibodies )
obstetric cases ) and testing the serum of the
to detect antigens on an
patient for ABO group PIGMENT PRODUCTION ON TSA
individual’s red cells
ABO Forward Typing antibodies.
Two Methods:
1. Slide Method REMEMBER:
Most commonly done because of ease of procedure If the expected results of both forward & reverse are not demonstrated,
Do not warm slides either a variation in the patient or technical error may exist!
Maximum observation time: 2 minutes
2. Tube Method
Preferred because this is more sensitive vs slide method
Do not perform above 20-24C EXPERIMENT 9
STAPHYLOCOCCUS
SLIDE COAGULASE TEST
ABO FORWARD TYPING
OBJECTIVES
Agglutinogens Designation of
Anti-A Anti-B Anti-H 1. Identify the different characteristics of Staphylococcus.
present blood group
2. Identify the differentiating characteristics of the pathogenic from
- - - None Bombay
non-pathogenic staphylococci.
- - + H O Rh +
MATERIALS
+ - + A, H A Rh +
1. Stock cultures of Staphylococcus aureus and Staphylococcus
- + + B, H B Rh + epidermidis.
+ + + A, B, H AB Rh + 2. Blood agar plates
3. Trypticase soy Agar (TSA)
ABO REVERSE TYPING 4. Mannitol agar
 2. From the same source, inoculate a BA plate by 4-quadrant 3. Incubate at 37⁰C for 24 hours.
S. aureus S. epidermidis
streaking and TSA slant by simple streaking. 4. After incubation, observe the color change in the medium.
TUBE COAGULASE TEST
Catalase Test
1. Add 1ml of hydrogen peroxide into the TSA slant.
2. Observe immediate appearance of gas bubbles – Staphylococcus
aureus & Staphylococcus epidermidis

QUESTIONS:

1. How can staph infections be prevented?

- Keep hands clean by frequent hand washing with soap and water
3. Incubate media at 37⁰C for 24 hours. (or alcohol)
MANNITOL FERMENTATION 4. After incubation, examine the isolated colonies on the ff: - Keep cuts and scrapes clean and covered with bandages until
S. aureus S. epidermidis a. Colonial characteristics of BA healed.
b. Hemolytic property on BA - Avoid contact with other people’s wounds or bandages.
c. Pigment production on TSA - Do not share personal items such as towels, clothing or cosmetics

Slide Coagulase Test 2. In relation to your profession as a nurse, when you inject medications
1. With a wax pencil, divide and label a clean glass slide as to patients, what should you do to avoid staphylococcal infection?
illustrated. - Proper disposal of used or contaminated materials used.
2. Place a small drop of NSS on the C (control) side and another - Aseptic technique. Clean client’s skin before administering
drop on the T (test) side. injection.
3. Make heavy suspensions in these drops of NSS with isolated
S. aureus S. epidermidis colonies growing on BA. 3. Is there a medication that you can give that can help in the
4. Add a loopful of plasma in the TEST and mix thoroughly with a eradication of the carrier state in a short term to minimize infections?
wire loop and observe for the appearance of clumping within 10 - Intranasal Mupirocin
seconds.
5. Compare the TEST with the saline CONTROL. 4. What is the catalase for?
CATALASE TEST
- It is used to differentiate staphylococcus from streptococcus
Tube Coagulase Test (catalase -).
1. Get a small test tube containing 0.5mL fresh human plasma
PROCEDURE 2. Get an inoculum and emulsify a visible portion of growth from
5. Why do you think staphylococci contain catalase? Why do they need
1. With the stock culture of Staphylococcus assigned to the group, isolated colonies in the plasma
that?
make a smear and do Gram stain. 3. Rub the material at the side of the tube while holding the tube in
- It protects the staphylococci from the accumulation of the toxic
an angle.
levels of hydrogen peroxide.
4. After, straighten the tube causing the plasma level to cover the
- In the metabolism of your staphylococci, their usually
site of inoculation.
aerobic/facultatively anaerobic – usually contain catalase
5. Incubate at 37⁰C and observe for clotting at intervals of 30
because in their metabolism it produces reactive oxygen species
minutes for 4 hours.
(e.g., hydrogen peroxide & superoxide). So, if they don’t have the
a. Observe the presence of gel or clot that cannot be
catalase, they can’t break hydrogen peroxide down, that’s why
suspended by just gentle shaking.
mamatay ang staphylococci.
b. If no clot forms after 4 hours – should be reintubated
overnight.
6. You mentioned that staph grows on BA and TSA or NA, so pwede ba
ibutang ang H2O2 (hydrogen peroxide) sa BA or dili?
Mannitol Fermentation Test
- No. The presence of H2O2 on the BA (which contains catalase)
1. Needed: Mannitol agar and TSA slant with the inoculum
might turn H2O2 into H2o and O2 rendering the result false
2. Get an inoculum form TSA using a sterile wire needle and stab
positive.
the mannitol agar until a few mm form the bottom.
7. How about in CA (chocolate agar)? 16. What are other coagulase variable staph? For example, tube 1. What is the common habitat of Staphylococcus?
- “okay ra” coagulase. So naa kay colonies na staph, sa plate catalase (+), so - Nasopharynx
imong gi subject to tube coagulase then result is (+) pero dili yellow - Skin
8. What would be our drug of choice for Methicillin-resistant iyang colonies. Would that signify that it is S. aureus or not?
Staphylococcus aureus (MRSA) since there is an increasing trend of - Yes gihapon bahalag dili siya beta-hemolytic pero nay species sa 2. Name 2 diseases commonly produced by S. aureus.
antibacterial resistance sa atong staph? staph na yellow pero negative siya sa tube coagulase - Staphylococcal Food Poisoning
- Vancomycin (Staphylococcus sciuri; Staphylococcus saprophyticus) - Scalded Skin Syndrome (SSS) aka Ritter’s Disease
- And also (Benozolin???)
17. Give an example of staph species na coagulase positive na dili S. 3. S. aureus can cause nosocomial infection. What is nosocomial
9. The sample used for coagulase test is plasma, differentiate plasma aureus. (found in dog bites) infection?
from serum. - Staphylococcus intermedius - Nosocomial infections are also known as hospital-acquired
- Plasma contains fibrinogen. Serum has no fibrinogen. infections because they can be acquired or contracted within the
- Serum is collected in a tube WITHOUT anti-coagulants while 18. Aside from coagulase, what type of control was used? hospital environment.
plasma is collected WITH anti-coagulants. That’s why fibrinogen - Negative control kay walay agglutination ni happen - Transmission via the healthcare workers, patients, hospital
is preserved in samples with anti-coagulants that prevent the equipment, or interventional procedures; direct contact to
clotting cascade to activate - wala siya naka clot. 19. It was mentioned that the slide coagulase test detects the cell-bound people, surfaces or objects within the hospital/clinical setting.
- Serum: red/yellow top (ang kanang lid sa test tube ba) coagulase, this reacting to your fibrinogen but in your tube coagulase
- Plasma: *wala natubag haha detects free or extracellular coagulase, reacting indirectly with the
fibrinogen. So, why indirect?
10. In the coagulase test, what coagulant should we avoid? - Formation of a complex of coagulase with the coagulase relating EXPERIMENT 10
- Citrate because it causes auto-clotting. factors (CRF) – modified or derived thrombin which react with NEISSERIAE
- Avoid citrated plasma because there are microorganisms that your fibrinogen or form the fibrin clot. OBJECTIVES – To observe the microscopic and cultural characteristics of
utilize citrate would produce false positive results. Neisseria
20. MRSA is an infection that is so hard to treat because the resistant siya MATERIALS
11. What sample did we use when we performed the coagulase test? to many antibiotics. As a nurse, it’s one of the feared infections to • Culture Neisseria meningitidis • Maltose agar
What anticoagulant was used? acquire. You were assigned to a patient that has pressure ulcer with • Culture N. gonorrhea • Sucrose agar
- EDTA or Ethylenediaminetetraacetic acid MRSA. The doctor ordered contact precaution, as a nurse what PPEs • Culture N. sicca • Lactose agar
are to be used? • Culture Branhamella catarrhalis • Oxidase reagent
12. When we look at the agar plate, what do we look for to say that - Gown and gloves
• Chocolate agar (CA) • Wire loops
pathogenic ang staph na gi isolate? - Dispose after use. Do not wear or use when with other patients.
• Nutrient agar (NA) • Wire needles
- Zone of hemolysis – beta id more pathogenic
• Glucose agar • Alcohol lamp
- Yellow pigmentation – the more pigmented the mor pathogenic Staphylococcus
BASIS Staphylococcus aureus PROCEDURE
the strain of staphylococcus is epidermidis
- Coagulase Gram Staining Gram (+); purple Gram (+); purple 1. When using Biosafety cabinet (2 or 3??? Di ko sure) – be careful.
Microscopic 2. Do gram staining of stock cultures
Staphylococci (clusters) Staphylococci (clusters) - Don’t forget to heat fix prior to staining.
13. Which is pathogenic? Morphology
- S. aureus Round, medium-sized, 3. 4-quadrant Streaking for NA and CA
Colonial Round, raised, opaque, - Streaking – method of spreading cultures onto surface of
circular, raised, opaque,
Appearance on circular, with soft agar
14. What is the cause of the yellow (golden) pigmentation of S. Aureus? smooth with a soft
BA consistency colonies - Before streaking another quadrant, wire loop must be
- Staphyloxanthin consistency colonies
Pigment Prod. Yellow White thoroughly incinerated to attain isolated colonies
REMEMBER!!! Hemolysis on BA Beta-hemolytic Non-hemolytic - Quadrant streaking - to fully exercise the spread of colonies
Au = Gold from each other thus attaining even more isolated ones
Slide Coagulase (+) clumping (-) clumping
Aureus = golden/yellow pigment - More quadrants = better isolation
Tube Coagulase (+) coagulation/clotting (-) coagulation/clotting
Especially samples that contain many organisms
Catalase (+) gas bubbles (+) gas bubbles
- Subsequent quadrants when streaked must touch the
15. For the tube coagulase, ngano mo check paman tag clots in intervals Mannitol Yellow;
Pink; (-) fermentation previous one a few times over to carry a minimal colonies
of 30 minutes? Why not incubate directly for 24 hours? fermentation (+) fermentation
for further spreading
- Some strains produce fibrinolysin that can dissolve the clot
 - Purpose – to attain isolated colonies on the last quadrants
Isolated colonies – individual colonies with clear
separation from neighboring colonies
When streaking, avoid “plowing” on the agar
surface
4. Perform same procedure for Chocolate Agar
5. Tube inoculation for glucose, maltose, sucrose and lactose
6. Inoculation is by stabbing ¾ to bottom
- Don’t forget to heat mouth of tubes prior to inoculation
7. Once done, plated agars and tubes must be incubating at 37⁰C Neisseria meningitidis
for 18-48 hours before reading the results.
8. After incubation, growth from plated agars are subjected to
Oxidase Testing.
- A presumptive test for Neisseria spp.
- Oxidase rgt us dropped onto the colonies resulting to a black
discoloration after a few seconds
- Oxidase rgt – 1% p-aminodimethylaniline oxalate
9. Tube Fermentation – differential testing

RESULTS Neisseria sicca & B. catarrhalis Neisseria sicca

ALL are Gram (-) bacteria

Branhamella catarrhalis

Neisseria gonorrhea

QUESTIONS
1. What is the normal flora of the rectum?
- Escherichia coli - enteric: Gram (-) bacilli
2. Do you think chocolate agar would be the best medium to
definitively grow our Neisseria gonorrhea or should we choose
another medium?
- Thayer Martin Agar
2.1 What does it contain?
- Vancomycin – inhibits gram (+) bacteria) esp., Staphylococci spp.
- Colistin – inhibits gram (-) bacilli
- Nystatin – inhibits yeast/fungi
3. Selective medium for N. gonorrhea and N. meningitis?
- Thayer Martin Agar
4. How does Thayer Martin Agar differ from the Modified TMA?
What is added?
- Addition of antibiotics/antimicrobial agents specifically,
trimethoprim lactate (babies)
5. For the transport of N. gonorrhea specimen, what should be done to
get the best results?
- Best to be placed in a transfer medium with high CO2 (can
survive longer) – when exposed to air, dies quicker
5.1 In a practical setting, how do you incorporate CO2? How do you
get an environment with sufficient CO2 levels?
- Place the sample in a closed container.
- In the laboratory, to get high CO2 levels, we light a candle then
cover it right away.
- Usage of the candle jar as a method of incubation
6. For N. gonorrhea, it doesn’t grow on Nutrient agar but it grows on
Chocolate agar. What is in CA that differs from NA? Why does it grow
on CA?
- CA has X-factor, V-factor, hemoglobin components and NAD. It
also has hemolytic blood cells (ruptured). di ko sure if sakto ako
nadungog hehe
7. It was mentioned that one of the clinical diseases brought about by N.
gonorrheae is Ophthalmia neonatorum. What does that imply
especially for us health care practitioners when we have expecting
mothers as our patient?
- Inform the mother of the condition and what it entails for her
child. As the bacteria has congregated in the vaginal canal,
doctors would opt for the mother to undergo a cesarean section
than NSVD to prevent the child to be in contact with the bacteria.
- If for example, we have an expecting mother who does not know
her status is, it is very important for the practitioners who’re
handling the case to have them tested for any STDs esp. knowing
that the infant can have ophthalmia neonatorum or any dss.
brought about by STDs from the mother.
8. Ex. The mother has full blown dse. of N. gonorrhea which has spread
to her blood system, and you want to get a blood culture. Would you
be using the same blood culture bottle as you have with other
infectious dss?
- No – N. gonorrhea is a fastidious microorganism
9. What is the component present in the standard/routine blood culture
bottle that inhibits Neisseria spp? Why can’t we use the regular blood
culture bottles when suspecting disseminated with coccal infection?
- It contains Sodium polyanethole sulfonate which inhibits N.
gonorrhea. We use a special blood culture bottle.
10. 40 y.o. Px complained of urethral discharge, suspecting N. gonorrhea.
How would you collect this sample?
- Urethral sample using a cotton swab or dacron tip/rayon tip
Ideal: Dacron tip/Rayon tip
If wala nagyuy lain: Cotton swabs to make it more viable,
add amies charcoal medium (microbial).
Cotton tip swabs contain toxic fatty acids inhibiting growth
of N. gonorrhea.
“instead of using swabs, we use wire loops sa lab” – Sir Lee
11. Presumptive tests for Neisseria spp?
- Oxidase test
12. Definitive test N. gonorrhea?
- Gram staining + culture
- Culture: not only growing bacteria on the agar plate but rather
doing work which includes Sugar Degradation Test & Oxidase
Test.
- In special cases, (male Px with penile discharge), it would suffice
to do Gram staining mainly because the sensitivity of Gram
staining is 90% with a specificity of 95%.
- Specimens from female Px, immediately proceed with laboratory
tests (culture and other work ups)
13. If child ang Px, what steps should you do? Should you stop at culture
or do you need other diagnostic modalities and why?
- Should order ALL – to ensure that there would be no
complications.
- Other modalities: Nucleic acid amplification test, serologic tests
(for IgM, IgA or IgG) depending on availability of institution
- N. gonorrhea in 4 years and below – has social implications which
is why we can’t release results basing on one test only
14. Sample needed for N. meningitidis?
- Cerebrospinal fluid (CSF) via lumbar puncture – insertion of
needle in between L3 and L4
15. What is the precaution done when collecting sample?
- Wearing of proper PPE – mask, gloves, gowns
- First job is to protect yourselves – may either infect your
coworkers or you families (be defensive)
- Consider everyone is infectious esp. if wala pay test
results/diagnosis but the clinical presentations of meningitis can
be very vague and very constitutional – can’t tell right away if its
meningitis
16. If wala gyud mo katarong ug PPE but you were handling a Px with (+)
meningitis, what should you do?
- ??? first, then give yourself a post-procedure prophylaxis along
with the other people you’ve been in contact with
- Meningitis: Px mental status deteriorates.
- Contact tracing is very important so can be given prophylaxis.
- Crede’s prophylaxis silver nitrate but sa hospital we use now
erythromycin
17. Why is it ang description sa (+) Oxidase Test is purple, why is it black sa
NA?
- It’s actually a spectrum of colors kay diba w/in seconds the color
changes from purple then mo black siya. Black is usually the end
color of the oxidase test. – Doc Cebrecus
*from other section
18. What causes color change in the fermentation test? What was used in
the sugar test?
 - Phenol red, same indicator used in the mannitol test BASIS N. gonorrhoeae N. meningitidis N. sicca B. catarrhalis - Yes, urethral swabs from males is a diagnostic test for Neisseria
In the presence of acid, yellow Gram stain & Gram (-) Gram (-) Gram (-) Gram (-) gonorrheae. For females, it can be diagnosed through the
morphology diplococci diplococci diplococci diplococci following tests: urine testing, endocervical (vaginal) swabs,
19. Do you agree the test is a sugar fermentation test? Small, grayish Medium, Large, endocervical nucleic acid amplification test (NAAT). Specimens
Large, non-
white, convex, smooth, round, brownish,
- No, it is a sugar utilization test but way sugar neutralizes is Colonial pigmented, for the diagnosis of Neisseria gonorrheae is collected from the
translucent, moist, gray to opaque,
through oxidation. That’s why no gas produced. morphology
shiny colonies white colonies;
wrinkled,
smooth, friable
urine, vaginal discharges, synovial fluid and etc. (ay mog salig sa
on CHOC coarse, dry akong answer kay di ko sure ani haha)
with smooth or encapsulated with a
20. Intercellular vs extracellular. agar and
irregular strains are “hockey puck”
adherent
- Intracellular microbes capable of growing and producing inside margins mucoid consistency 6. In cultivation of Pathogenic Neisseria, selective media are employed
host cell. Colonial
Breadcrum Round, opaque,
aside from routine media. Compare and supply the differences in
morphology these media:
Not applicable Not applicable bs-like convex and
21. What cell was shown on the slide? on Nutrient
colonies greyish white
Agar
- Neutrophils (mostly) and macrophages Thayer Martin Modified Thayer Martin Martin Lewis Medium
Oxidase
(+) (+) (+) (+) Medium Medium
Test (+/-)
22. Implication of finding intracellular and extracellular diplococci, does it Category Selective and Selective and enriched Selective and enriched
Glucose
matter? Let’s say you have a Px with gonorrhea urethral discharge, I A A A NC enriched
(A/NC)
Medium Vancomycin, Neisseria gonorrheae; Neisseria gonorrheae;
got pus smeared, I saw an intracellular gram (-) diplococcus, what Maltose
NC A A NC is used Colistin and Vancomycin, Colistin, Vancomycin, Colistin,
would it imply? (A/NC)
for: Nystatin Trimethoprim, Nystatin Trimethoprim (Proteus
- Intracellular diplococci indicate ACTIVE diseases. If extracellular Sucrose
NC NC A NC spp.), Anisomycin
less active ang disease, so asymptomatic lang ang Px. (A/NC)
Lactose
NC NC NC NC
23. Why does each quadrant have different growth? (A/NC)
EXPERIMENT 11
- If there is growth on one quadrant, more isolated on the fourth
1. What is the mode of transmission for Pathogenic Neisseria? IDENTIFICATION OF ENTERIC ORGANISMS
quadrant so mas distinct ang colonies
- Neisseria gonorrheae is transmitted via sexual contact; may also LEARNING OBJECTIVES:
be spread from mother to newborn through birth. 1. To explain the importance of the different media used in the
24. What it the definitive diagnosis of Neisseria gonorrhea?
- Neisseria meningitidis is transmitted via droplet spray usually in isolation and identification of enteric organisms; and
- Carbohydrate utilization
close contact with others. 2. To identify the enteric bacilli based on their biochemical
- culture alone would not suffice in definitive diagnosis, such as in
properties
children (bc social discomfort) but can be serology tests, more
2. What are the two (2) species of Neisseria that are not of normal flora MATERIALS:
than one test
of the upper respiratory tract? 1. Eosine methylene Blue Agar
- For the Sugar test, refer to it as “carbohydrate degradation test”
- Neisseria gonorrheae – found in the genitalia, anorectal area, 2. Salmonella-Shigella Agar
(DO NOT use fermentation)
oropharynx or conjunctiva at time of infection 3. Bisthmus Sulfate Agar
25. Common disorder of N. gonorrhea? - Neisseria meningitidis – colonizes the oropharyngeal and the
- STD with samples collected via urethral swab, urine nasopharyngeal mucous membranes
sample/collection
3. What do you call a collective term for the majority of non-pathogenic
26. N. Meningitidis: bacterial meningitis mode of transmission? Neisseria?
- Droplet spray - Commensal Neisseria.

27. Reagent? 4. What kind of transport medium used when coupled with Cotton
- 1% tetramethyldihydrochloride(?) swabs upon collection when bedside inoculation cannot be
performed?
28. What if you inoculate using a nichrome wire, is this possible? - Amies Transport Medium added with charcoal.
To detect biochemical reactions:
- No bc might give false positive reactions. Instead use swabs or
5. (Yes or No) Intracellular gram (-) diplococci when seen in urethral 1. Triple Sugar Iron Agar (TSI) - For Triple Sugar Iron Agar reaction
platinum loop
swabs from males is diagnostic for infection of Neisseria 2. Lysine Iron Agar (LIA) - For Lysine Decarboxylation & Deamination
gonorrhoeae? What about for female patients? 3. Motility-Indole-Ornithine Agar (MIO) - For Motility-Ornithine
Decarboxylation
 4. Tryptone Broth - For Indole Production EOSIN METHYLENE BLUE
5. Simmon’s Citrate Agar (SCA) - For Citrate Utilization test MILDLY SELECTIVE for Gram - negative bacteria. Results:
6. Phenylalanine Agar - For Phenylalanine Deamination • This is because it contains dyes (Eosin Y & Methylene Blue) • Salmonella will not ferment
7. Urea Broth - For Urease test that are toxic to Gram-positive bacteria lactose, but produce hydrogen
8. PAP & PAF Agars - For pigment production tests DIFFERENTIAL - for fecal coliform bacteria sulfide H2S gas. The resulting
• allows you to distinguish (just by the way it looks) coliform colonies will appear colorless
COLIFORMS bacteria from non-coliform bacteria. with black centers.
Morphology: • Shigella do not ferment lactose
- rod-shaped, Gram-negative, non-sporeforming bacteria METALLIC GREEN SHEEN or produce H2S so the resulting
- may be motile or non-motile - present due to the interaction with the dyes and strong acid-end colonies will be colorless Salmonella
Lactose Fermenters: products of fermentation in • Coliform bacteria, such as the E.
- lactose is found in the EMB media specific types of bacteria coli will ferment the lactose in
- When they ferment lactose, they produce acid and gas - The presence of a flagella can also the media, resulting in bacterial
byproducts. contribute to the metallic sheen, growth with a pink color. They
Location: most commonly: do not produce any H2S.
- usually found in the gut, many are not harmful to humans • E. coli • Enterobacter and Klebsiella
- when found in food or water, it means that it has been
• some species of Citrobacter appears larger than E. coli,
contaminated with fecal material
spp. mucoid, pale, opaque, cream to
• Coliforms ferment lactose • some species of Enterobacter pink Shigella

• Produce acid and gas by products spp

• Ph is lowered (low = dark purple) Limitations:
• Forms a dark purple center (nucleated) • It is recommended that
SALMONELLA - SHIGELLA AGAR biochemical, immunological,
Culture Media for the Isolation of Enteric Bacteria Moderately differential for the isolation, cultivation, and differentiation of molecular, or mass
Color of Colonies Salmonella spp and some Shigella spp. spectrometry testing be
Classification

• Is a modification of the Deoxycholate Citrate Agar performed on colonies from

of Medium
Indicators

Inhibitors
Medium

Sugar/s

Fermenter

Fermenter

• Is recommended for testing clinical specimens and food testing pure culture for complete
Lactose

Lactose
Non-

for the presence of Salmonella spp and some Shigella spp. identification.
Principle: • The incorporation of brilliant Coliform bacteria
• The inclusion of bile salts, sodium citrate, and brilliant green green into this medium makes
Colorless (takes

Mildly Selective
greenish sheen

it highly selective and has been shown to inhibit the growth of

up the color of
colonies, i.e.,

the medium)

serve to inhibit Gram-positive, coliform organisms and inhibit

pink or with
Blue (EMB)

Differential
Methylene

Methylene

Methylene
Lactose,

some Shigella.
Colored
Sucrose

Eosin Y,

Eosin Y,

swarming Proteus spp while allowing Salmonella spp to grow

Eosin

Blue

Blue

• Beef extract, enzymatic digest of casein, and enzymatic digest of • The bile salts may crystallize over time. They appear as small
animal tissue provide sources of nitrogen, carbon, and vitamins spider-like puff balls within the medium and do not affect the
required for organism growth performance of the medium.
• Thiosulfate and ferric citrate permit the detection of hydrogen • Some strains of Shigella such as Sonnei and dysenteriae serovear
Colorless (takes
Sodium Citrate,
Brilliant Green,

up the color of
Ferric Citrate,
Shigella Agar

the medium)
Neutral Red,

Moderately

1, may ferment lactose relatively slowly, and colonies change to

Differential
Salmonella

Thiosulfate

sulfide by the production of colonies with black centers.

Bile Salts

Selective
Lactose

Sodium
(SSA)

lactose-fermenting after cultivation for 2 or more days.

Pink

• Neutral red turns red in the presence of an acidic pH, thus

showing fermentation has occurred • A few nonpathogenic organisms may grow on SSA
Uses:
Bismuth Sulfite Agar (BSA)
• Used as a selective and differential medium for the isolation of
- A modification of the original Wilson and Blair selective diagnostic
Ferrous Sulfate;

Highly Selective
Bismuth Sulfite

Bismuth Sulfite

Brilliant Green

for Salmonella
Black colonies

Salmonella and some shigella spp from clinical and non-clinical

with metallic
Agar (BSA)

medium used for the isolation of Salmonella typhi and other

Inhibited
Glucose

specimens
sheen

typhi

Salmonellae from food and other materials

• This is not recommended for the primary isolation of Shigella - Classification: Highly Selective
• It was developed to aid in the differentiation of lactose and non-
lactose fermenters from clinical specimens, suspected foods, and
other such samples.
Composition and principle ORGANISM EMB SSA BSA Procedure
• Peptone Mostly inhibited, - Single well-isolated colony for inoculation
Escherichia Dark-centered with Red to pink;
• HM Peptone B coli greenish metallic colorless with a
dark brown, green - With a wire needle, inoculate the TSI by making a line on the slant
surface, no metallic surface, then stab the butt until a few mm from the bottom, then
• Dextrose (Glucose): carbon source sheen pink center
sheen streak the surface of the slant
• Disodium phosphate: maintains the osmotic equilibrium - Incubate at 37ºC for 18-24 hours
Similar to E. coli but
• Ferrous sulfate: aids in the detection of hydrogen sulfide Enterobacter/
somewhat larger;
White or cream
Raised, mucoid,
production and the formation of ferrous sulfide - an insoluble Serratia with pink center,
often “fish-eyed” in silvery sheen Reading and Interpretation
black precipitate that gives Salmonella colonies their opaque mucoid
appearance A/A - Yellow slant, yellow butt
characteristic brown-black coloration - Dextrose fermented, lactose and/or
• Agar Larger than E. coli,
Red to pink; sucrose fermented
Klebsiella mucoid, tend to
• Bismuth Sulfite coalesce, often
colorless with a Mostly inhibited Alk/A - Red slant, yellow butt
pink center
• Brilliant green convex - Dextrose fermented, lactose and/or
sucrose not fermented
Procedure Green; black (H2S Alk/Alk or Alk/NC - Red slant, red/orange
Proteus Translucent, Black-centered,
producer), mostly butt
1. Suspend required amount of dry powder in 1000 ml distilled water. colorless, swarming clear periphery
inhibited
2. Heat to boiling to dissolve the medium completely. - None of the 3 sugars are fermented
Mostly inhibited,
3. Mix well to disperse suspension and pour thick plates (25 ml medium Pseudomonas Translucent,
translucent, Inhibited An H2S producing organism may produce so much black
per plate). colorless
colorless colonies precipitate (Ferrous Sulfide) that the acidity in the butt
4. Allow the medium to solidify with the dish uncovered.
S. typhi black with is completely masked
5. Dry the plates before use Opaque,
sheen or dotted However, if H2S is produced, an acid condition does
6. Inoculate the plates with the Salmonella Translucent,
transparent,
black or greenish exist in the butt even if not observable and should be
specimen. uncolored; black-
colorless gray; other recorded as such
centered, clear
7. Incubate inoculated media periphery
Salmonella are black
for 48 hours at 35 degrees or green
Gas production is indicated by:
8. Examine after 24 hours for Mostly inhibited, - Splitting of the medium
typical colonies. Opaque, S. flexneri and - Single gas bubble
Shigella Translucent,
• Salmonella typhi - colorless
transparent, S. sonnei are brown, - Complete displacement of medium from bottom of
Good-luxuriant growth; colorless raised, and tube
Black with a metallic crater-like - Slight indentation of the medium from side of tube
sheen
BIOCHEMICAL REACTIONS LYSINE DECARBOXYLATION AND DEAMINATION
Basis of Reactions and Identification Medium and Principle
- Metabolic action of microorganism on different media to produce a - Lysine Iron Agar (LIA)
specific chemical change which can then be detected by the addition - Determines whether the organism can decarboxylate or deaminate
of chemical agents lysine
- Microorganisms are then identified on the basis of their metabolic - Decarboxylation of lysine produces cadaverine
activity on these different media after an incubation period of 24 to 48 - Indicator: Bromcresol Purple
hours
Procedure
SUGAR FERMENTATION – HYDROGEN SULFIDE PRODUCTION - Using a straight wire needle, inoculate the LIA by stabbing the butt
Medium and Principle twice all the way down, then streak the slant surface
- Triple Sugar Iron (TSI) agar - Incubate at 37ºC for 18-24 hours
- Determines ability of organism to attack a specific
carbohydrate, with or without production of gas
- May also determine possible hydrogen sulfide production
- Presence of cysteinase
- Indicator: Phenol Red
Reading and Interpretation: Procedure
P/P - Purple slant, purple butt For Indole Production - Streak the slant surface of the Phenylalanine agar with the test
- Absence of lysine deaminase, presence of - Add a few drops of Kovac’s reagent organism
lysine decarboxylase Indole (+) - Incubate at 37ºC for 24 hours
- Lysine was not deaminated but was - Pink color is observed - Add 5 drops of FeCl3 after incubation
decarboxylated - Presence of tryptophanase
- Cadaverine was produced Indole (-) Reading and Interpretation
- Yellow color is observed - Positive - Green slant
P/Y - Purple slant, yellow butt - Absence of tryptophanase - Presence of phenylalanine deaminase
- Absence of both lysine deaminase and - Phenylalanine was deaminated
decarboxylase For Ornithine Decarboxylation - Phenylpyruvic acid was produced
OD (+) - Negative - No change in color of slant
R/Y - Red slant, yellow butt - Purple color throughout the tube or 2/3 of the medium from the
- Presence of lysine deaminase, absence of lysine decarboxylase top is purple UREASE TEST
- Black color in the butt or along the stab line indicates hydrogen sulfide - Putrescine was produced Medium and Principle
production OD (-) - Urea broth
- Splitting of the agar indicates gas production - Bottom of medium will be bright yellow with a narrow rim of - Some bacteria possess the ability to split urea into ammonia and
purple at the top carbon dioxide
MOTILITY-INDOLE PRODUCTION - ORNITHINE DECARBOXYLATION - Putrescine was not produced - Enzyme: Urease
Medium and Principle - Done for the rapid identification of Proteus organisms
- The ability of an organism to give a positive indole test depends on CITRATE UTILIZATION - Indicator: Phenol Red
whether or not it possesses tryptophanase Medium and Principle
- Reagent: Kovac’s Reagent (5 drops) - Simmon’s Citrate Agar (SCA) Procedure
- MIO medium be tubed at least 2 inches to provide anaerobic - Determine if an organism is capable of utilizing citrate and an - Inoculate a heavy loopful of the test organism from the TSI into the
conditions necessary for the detection of ornithine decarboxylase ammonium salt as the sole source of carbon and nitrogen respectively urea broth
activity for metabolism, with resulting alkalinity - Incubate at 37ºC for 24 hours
- Positive test - Production of alkaline putrescine - Indicator: Bromthymol Blue - Observe for color change
- This test is only valid for organisms which ferment dextrose
- Indicator: Bromcresol Purple Procedure Reading and Interpretation
- Inoculate the surface of SCA by streaking one loopful of the test - Positive - Medium turns into a dark pink color
Procedure organism - Presence of urease
- Stab the MIO medium once with a straight wire needed to the bottom - Incubate at 37ºC for 48 hours - Ammonia and carbon dioxide were produced
of the tube - Negative - No change in color of medium
- Incubate at 37ºC for 24 hours Reading and Interpretation
- Read the motility and decarboxylase reactions before adding Kovac’s Positive PIGMENT PRODUCTION
reagent - Growth is accompanied by color change of Medium
indicator; changes slant from original green to For Pseudomonas aeruginosa
Reading and Interpretation deep Prussian blue color - Pseudomonas agar P (PAP) or King’s A medium
For Motility - Citrate was utilized - Pseudomonas agar F (PAF) or King’s B medium
Positive (Motile) - Presence of citratase For Serratia marcescens
- Motile organisms migrate from the stab line - Sodium bicarbonate and ammonia were produced - PAP
and diffuse into the medium causing Negative - Agar remains green
haziness; may exhibit fuzzy streaks of growth Procedure
- Presence of flagella PHENYLALANINE DEAMINATION For Pseudomonas
Negative (Nonmotile) Medium and Principle - Inoculate PAP and PAF and incubate at 37ºC for 24H
- Bacterial growth accentuated along stab line; - Phenylalanine agar For Serratia
surrounding medium remains clear - Determines the ability of the organism to deaminate phenylalanine to - Inoculate PAP and incubate at room temperature for 24H
- Absence of flagella phenylpyruvic acid by enzymatic activity with resulting acidity
- Reagent: Ferric Chloride (FeCl3)
 Reading and Interpretation
For Pseudomonas – Positive
- PAP - Pyocyanin is produced
- PAF - Pyoverdin or
Fluorescein is produced
For Serratia – Positive
- Prodigiosin is produced
Summary of Biochemical Reactions
Plated
Colonial TSI
Organism Mediu
Morphology Slant Butt H S
LIA
m 2
ANSWER: Escherichia coli is the predominant coliform in the large intestine
of humans and women are predisposed to UTIs because of their short
urethras and because of the close proximity between the anus and
vagina/urethra.
4. Are the Enterobacteriaceae pathogenic when isolated outside the
gastrointestinal tract?
ANSWER: Yes. Most of the genera of the family Enterobacteriaceae are
part of the normal flora of the intestinal tract and are considered
opportunistic pathogens. They act as opportunistic pathogens when they
are introduced or isolated in body locations where they are not normally
M I O SCA PAD UREA PAP PAF found (e.g., normally sterile sites such as the blood, CSF, etc.), especially if

Colonies the host is immunosuppressed or debilitated.

with
E. coli EMB greenish A AG - P/P + + - - - - - - QUESTIONING:
metallic 1. What’s the physical state of your MIO?
sheen - Semi-solid
Jet black
colonies 2. Give the semi-solid state, what are we able to appreciate in this
Salmonella
BSA with Alk A + P/P + - - - - - - -
typhi tube/medium? The reason why its semi-solid?
metallic
sheen
- To Identify any turbidity or swarming around the line of
Colorless stabbing
Proteus
vulgaris
EMB swarming Alk AG + R/Y + + - - + + - -
colonies 3. What other tests can we do with the MIO tube?
E. Fish-eye - Indole production with the use of Kovac’s reagent
EMB A AG - P/P + - + + - - - -
aerogenes colonies (+) Indole production = RED
Colorless - Ornithine Decarboxylation – checks the presence and
P. + +
aeruginosa
EMB translucent Alk Alk - P/P + - + + - - absence of ornithine decarboxylase
colonies *if wa nadaw ka kahibaw if unsay enzyme sa test kay check nalang the name – ez radaw
Colorless lol
S.
dysenteriae
SSA translucent Alk A - P/Y - - - - - - - -
colonies 4. Color change for MIO tube – end product?
K. Large - Diamine putrescine
pneumonia EMB mucoid pink A AG - P/P - - - + - + - -
e colonies 5. You see a purple rim at the top of the MIO tube – positive test or not?
Colorless
Serratia - Negative – purple coloration throughout the medium not only
marcescens
EMB translucent Alk A - P/P + - + + - - + -
colonies
the rim
- Should be at least half or 2/3 down - violet
QUESTIONS:
1. Give 2 enteric bacteria in this experiment that are pathogenic when
6. When you say throughout the medium, what if I have a yellow tinge
isolated from stools.
towards the butt of the medium. Should I consider it negative or
ANSWER: Shigella dysenteriae and Salmonella typhi
positive?
- Yes???
2. What is the significance of Escherichia coli when isolated from the water 8. Simmon’s Citrate Agar – enzyme & indicator?
- Due to glucose fermentation (producing acid)
supply? - Enzyme: citrase
ANSWER: It means that the water supply is contaminated with fecal matter - Indicator: Bromthymol Blue
7. Ornithine Decarboxylation?
and is not safe for consumption. - Positive test: Prussian Blue
- Indicator: Bromcresol Purple
- Positive test: Purple
3. Why is Escherichia coli the most common cause of urinary tract infection *color of indicator kay positive of your test (clue daw pero not always, u g?)
especially in females?
9. End products responsible for color change? 19. If colorless daw ni grow sa MacConkey – what are the results? 31. Health education for E. coli?
- Ammonia and sodium bicarbonate – making the alkalinity of the - Alk/A with (+) H2S for Salmonella - Proper wiping after defecation from front to back – prevent bacteria
medium - Alk/A with (-) H2S for Shigella dragged to urethra

10. What other clues would signify the test as positive? 20. Ex. Colorless imo colonies sa MacConkey nya pag culture to TSI kay Reminders:
- Growth of organism A/A. Pwede bah? -ella (species): “princess” – they don’t move (non-motile); clear in MIO
- If the microbe inoculated in citrate tube will not be able to grow in the - Dapat Alkaline sa TSI Except for salmonella
medium as well??? - If mo reach out ang color sa agar – it means the nutrients sa agar kay
wala na (colored to colorless) A/A – EKE (E. coli, Klebsiella, E. aerogenes)
11. Prussian blue interpretation? (+) Citrate Test - Slow phenomenon – slow _____ ??? (di kos sure)
- Citrate was utilized??? Orthenine – purple – automatic daw E. aerogenes
- Presence of citratase 21. What organism is lacking if slow lactose fermenters sila? What test can
- Sodium bicarbonate and ammonia produced be used to distinguish slow and fast?
- Permease EXPERIMENT 12
12. EX. We have patho- & non-pathogenic enterobacterciae, mostly sa DISTRIBUTION OF FUNGI IN THE ENVIRONMENT
tool culture, nya mo pass kag non-sterile, clean container with stool 22. What enzyme is present in both slow and rapid?
- Accept kay basta clean ok na - Galactosidase (lactose – glucose and galactose) OBJECTIVES
- Describe the general distribution of fungi in the environment
13. Upon culture, selective/differential imo medium, so bacteria in the 23. PH Indicator for TSI? - Explain how to identify fungi
container will be inhibited but what should I look for after culture? - Phenol Red
Descriptions – pathogenic organisms. MATERIALS
- MacConkey Agar – colorless with pink colonies 24. Of the organisms (excl. Pseudomonas), incubate for 6-8H, what do I 1. Saubouraud’s Dextrose agar (SDA)
- Shigella and salmonella – translucent with slightly black due to expect to read sa TSI? Usually incubated at 18-24H pero feel lang nimo 2. Cotton swab
interaction with H2S in SSA mo check at 6-8. 3. Sterile Normal Saline Solution (NSS)
*doh di nako sure sa kalibutan - Glucose can be fermented in 6-8H 4. Lactophenol Blue
- Shigella – fried egg appearance; no H2S - Cannot yet distinguish organisms 5. Glass slide and cover slips
- Salmonella – has H2S; colorless with black center 6. Scalpel blade
25. Proteus spp. – Alk/A siya but sometimes A/A ang readings sa TSI.
14. Preliminary tests - Salmonella within what medium? Why? – Note: non-lactose fermenter siya PROCEDURE
- TSI and LIA - Some strains of Proteus vulgaris can ferment sucrose 1. Moisten a sterile swab in sterile NSS and swab the area
- After 18-24H - glucose, lactose and/or sucrose is fermented (countertop).
15. Alk/A with H2S imo TSI then LIA is R/Y – organism? 2. Streak over the surface of the SDA plate the cotton swab using
- Proteus vulgaris 26. There are black precipitates in your TSI tubes, what do you call those? simple streaking method.
- Ferrous sulfide 3. Incubate the plate at room temperature right side up.
16. Ilabay siya or e further follow-up/identify? – stool culture 4. Observe the growth of the fungi and take note of the ff:
- Non-pathogenic in stool – ilabay 27. What gas does it produce? a. Type of colony
- Hydrogen sulfide b. Nature and consistency of growth
17. Ex. E. coli, pathogenic mani siya. Unsa man na preliminary tests ang c. Color of growth (surface and reverse of plate)
buhaton (2)? 28. Reading – pink slant with black butt
- LIA and MIO - Alk/A with H2S – H2S only in acidic medium Wet Mount Preparation
- Black = acid 1. Obtain a thin portion of agar and colony using a scalpel blade.
18. LIA is P/P nya MIO is Motile, Indole (-) and Ornithine (+). Would that 2. Remove the cut portion and transfer to a clean glass slide.
narrow down sa imong E. coli? 29. Where can we appreciate ferrous sulfide formation? 3. Add a drop of Lactophenol Blue and put on a cover slip.
- TSI and MIO daw - LIA 4. Observe the ff: (LPO and HPO)
- TSI – A/A a. Nature and type of mycelia
- If indole (+) – needs further examination for E. coli 30. UTI – organism? b. Characteristic arrangement, size, shape and type of
*sa CVGH Lab daw kay if 2 yrs old and below kay they examine the child for E. coli – both colored
- E. coli spores formed
and colorless colonies
RESULTS Sporangium Conidia
Macroscopic
Sporangiospore Phialides
(gross) Aspergillus
Rhizopus

Vesicle

Sporangiophore Conidiophore

surface reverse Rhizoids

Cottony to fluffy, white to yellow, Buttery, translucent, with rugose
becoming dark gray colonies Gross Appearance:
Grayish and loose Gross Appearance:
mycelium dotted with Powdery with sulfur
Microscopic
brown sporangia yellow areas scattered
spores
over the surface
sporangia
Spores SUPPLEMENTAL NOTES (refer to illustrations above)
Mucor
- all colonies are on SDA incubated at room temperature
Sporangia
RHIZOPUS SPECIES
sporangiophore
Sporangiophore Gross Appearance
growing (2-4 days) coarse, wooly colony which soon fills the test tube
with a loose, grayish mycelium dotted with brown or black sporangia
Penicillium
Conidia Microscopic appearance
Phialides Large, broad, nonseptate, hyaline mycelium that produces horizontal
runners (stolons) which attach at contact points (medium or glass) by
Metulae rootlike structures called rhizoids
From these contact points arise clusters of long stalks called
sporangiosphores
Ends are terminated in large, round, dark walled sporangia (spore
sacs)
Gross Appearance:
White, fluffy mycelium MUCOR SPECIES
Gross Appearance
Rapidly growing colony that fills the test tube with a white fluffy
mycelium, becoming gray to brown with age

Microscopic Appearance
Nonseptate, colorless mycelium without rhizoids
Gross Appearance:
Very powdery, whitish
 Sporangiosphores arise singly from stolons and branch profusely with - Colonies that are shiny and wax-like, mucoid, with a smooth QUESTIONS
sporangia containing many spores arising from the apex of each surface 1. What acid is present in the lactophenol cotton blue that
branch - No aerial hyphae are observed preserves fungal structures (one of the active components of
- Yeasts typically fall into this category lactophenol blue that also serves as a preservative)
ASPERGILLUS SPECIES - Moist, creamy, buttery, and some have a “bread-like” odor A: lactic acid
Gross Appearance
Grows rapidly (2-5 days) and appears first as a flat, white, filamentous COLONY MORPHOLOGY 2. Why not put the agar upside down?
growth which rapidly becomes blue-green and powdery with sulfur- Includes the topography or structure and A: this is to allow condensation because fungi grows faster in a moist
yellow areas scattered over the surface shape of the fungal colonies on the agar environment
surface and reverse, as well as the color
Microscopic of the colonies 3. Cotton blue is an acid dye that stains what substance present in
Branching septate hyphae, some of which terminally bear a REMEMBER: best to be as specific as the cell walls of fungi?
conidiosphore that extends into a large, inverted, flask-shaped possible when describing colonies A: Chitin (fibrous substance)
vesicle (sac) covered with small phialides that occurs only in a single
row and around the upper half of the vesicle 1. Rugose Colonies 4. How does fungi reproduce?
Long chains of small (2-3 um in diameter), spherical, rough-walled, - Colonies that are wrinkled or folded A: they produce sexually or asexually depending if they are perfect fungi or
green conidia form a columnar mass on the vesicle in appearance and are striated imperfect fungi.
Perfect fungi - reproduce sexually
PENICILLIUM SPECIES 2. Verrucose Colonies Imperfect fungi - reproduce asexually by budding or spore
Gross - Colonies that are mountainous, production
Growth is rapid, producing at first white colony which becomes wrinkled or convoluted in appearance
bluish-green and very powdery and striated 5. What are the necessary conditions for the growth of fungus?
A: Temperature - 25-30 degrees Celsius
Microscopic 3. Umbonate colonies Humidity - 40-50% relative humidity which is achieved by placing an open
Hyphae are hyaline and septate - Colonies that come to a peak or slight can of water inside the incubator
produce brushlike conidiosophores which exhibits branching metulae point/ elevation in the middle and
which is where phialides producing chains of conidia arise are button-like (heaped) 6. What media is used for fungal growth?
- There may also be a deep rugose A: SDA Is used because it contains peptones which provides a nutritious
COLONY TEXTURE furrows surrounding the button source of amino acids and nitrogenous compounds for the growth of fungi
Height of the aerial hyphae (what you can see above the surface of and yeast and this is ACIDIC
the agar) *doc ceb
What makes molds appear “fuzzy’ - final pH of SDA is acidic (5.6) but it is not the peptone that is
responsible for the acidity because peptones yield amine which
1. Cottony/Wooly are alkaline
- Colonies with very high, dense aerial mycelium that appears like a - dextrose is responsible for the acidity because fermentation of
sheep’s wool sugar yields acid
- HOW TO DESCRIBE: Fluffy, cottony, cotton-candy like (do not use - THERE IS MORE DEXTROSE/ GLUCOSE IN SDA THAN PEPTONE
any other descriptions)
- Colonies are fluffy, and sometimes quickly fill the plate if they are 7. what is the function of our cotton blue for the lactophenol cotton
rapid growers blue as one of the active components
2. Velvety A: gives color of the structure of the fungus
- Colonies whose aerial hyphae are lower and resemble velvet or
velour fabrics 8. it is one of the active components of lactophenol cotton blue that
3. Granular/Sugary acts as killing agent
- Colonies that are grainy-like granulated sugar or powder A: phenol (kills any viable cells)
- Aerial hyphae are flat, dry, rough, crumbly, or flour-lik
- Sometimes these colonies contain granules 9. why is it necessary to examine the surface and reverse side of the
4. Glabrous/Waxy plate? For our SDA
A: it is important to observe both sides of the plate for mold growth and
characteristics, because hyphae are either VEGETATIVE or AERIAL.
Aerial hyphae
- may support other structures such as: conidia, which
productive structures
- responsible for distinct characteristics , color and texture of
the front side of the colony
- extending above
Vegetative hyphae
- responsible for the characteristics of the reverse side
- food absorbing portion of the hyphae mycelium which is
under the agar surface
10. why do we incubate the plate at room temperature?
A: if we incubate at 37 then other bacteria would grow if we use SDA nga
plain
11. At what temp do we usually incubate pathogenic bacteria?
A: 37 degrees celcius
5. SDA Sabouraud dextrose agar, is a general purpose medium for the
growth of the fungi, what can you add to the SDA in order to grow
our organism of interest?
Since I want to grow slow growing fungi to prevent the overgrowth of
rapidly growing fungi, what should I add
A: Cycloheximide
12. what antibiotic is added to inhibit the growth of Gram negative
bacteria
A: Gentamycin (-----------------)
13. for the SDA, what provides for the nitrogen and vitamins required
for the orgs growth?
A: peptone
14. for our demonstration, what is the main differentiation between
our mucor and rhizopus species?
A: The main difference between Mucor and Rhizopus is that”
Mucor - does not have rhizoids and stolons
Rhizopus - has both rhizoids and stolons
Stolons = used for propagation
15. How do you describe a stolon?
- Horizontal stem (know parts kay para practicals)
16. How about for Rhizoids?
- Rootlike structures, usually for support
17. what are the 2 growth forms for our fungi?
A: Yeast - ovoid shape
- sometimes bud and fail to break off from their parent cell
(constrictions) they form a pseudohyphae (evident in Candida;
also described as chains or links of sausages)
- hyphae (individual structure; may be septated or nonseptated)
- mycelium ( matted hyphae)
Molds - growth of mycelium in agar; colony growth in agar; visible;
18. How does yeast appear under the microscope?
A: unicellular, bud like appearance
19. What do you call these yeast if they are unable to detach from
parent cells, diba they usually reproduce by budding
A: Psuedohyphae ( that is our clue, if naa gae constrictions from one cell to
the other then that is pseudohyphae)
20. 2 types of molds
A: Aerial - reproductive
Vegetative – gets the nutrients from the agar
21. how can we diagnose or identify the fungi or what are the 3
parameters that we have to take into consideration?
3 parameters that would aid us as to which specific fungi we are handling
1. Microscopic examination (most definitive)
2. Macroscopic appearance (supplements micro exam)
3. Growth rate
22. The minimum number of days for fungal cultures that we have to
incubate it before we say its negative
A: 21 days
- sometimes until 30 days para sure, para when we report it as
negative, it really is negative wherein no fungal elements have
grown in the agar
Then we can further subclassify our fungi into 3
Slow growers – 11 to 21 days
Intermediate growers- 6 to 10 days
Rapid growers – 5 days of less
22. how often do we have to check our agar or culture
A: 2-3 times a week - Most of fungi are opportunistic
23. what are the components/ reagents of lactophenol cotton blue
(LPCB) and purpose of each (IMPORTANT ANA SI DOC CEB)
Phenol- kills the live orgs
Lactic acid- preserves morphology of fungus/ fungal structure;
preservatice
Cotton blue- stains the chitin on the fungal cell walls
24. in the video, we used a scalpel , we made a cut and we got the
agar with the aerial mycelium and placed it in the wet mount,
why man?
A: to include vegetative mycelium or to appreciate both forms
More important: to minimize disturbing the morphology
- sometimes we can get our colonies using adhesive tape which
disturbs the morphology
25. What is the purpose of KOH and why is it the most common one
that we employ as a diagnostic method?
A: KOH is a clearing/cleaning agent. It clears/cleans away the living tissues
and leaves behind the resistant and refractory cell walls of our yeast cells
or fungal cells. Commonly order KOH especially if our first impression is a
fungal infection, esp those in the skin. We have to eliminate the tissues
which are not of interest and leave behind the fungal elements.
26. What is the difference between rhizopus and mucor?
A: Mucor - does not have rhizoids and stolons
Rhizopus - has both rhizoids and stolons
27. Give a part of rhizopus and mucor that is common to both.
A: both have sporangium which are found at the top of the sporangiophore
28. Why is strong alkaline needed for KOH?
A: KOH - potassium hydroxide; dissolves healthy skin cells leaving only the
fungal cells; CLEARING AGENT that is why it allows the fungi to stand out
more from the background because KOH dissolves other cells
 29. What is the most common class of antifungal drug? Q: do they have a hyaline EXPERIMENT 13
A: the -azoles (cream or OTC; Nizoral, Ketoconazole, antidandruff; A: TRUE SYSTEMIC MYCOSES
Fluconazole is the most common)
KNOW THE STRUCTURES!!! And definition ana si doc ilista daw; basin mugawas sa practical
Materials
1. Cultures of Candida albicans and Cryptococcus neoformans
TRUE or FALSE
2. Sabouraud’s Dextrose Agar (SDA)
Q: is fungi saprophytic (obtaining nourishment from the products of
- Nutritionally deficient
organic breakdown) in nature?
- has peptone which is used as a nitrogen and vitamin source that is
A: TRUE
needed for the bacterial growth
Q: Are they prokaryotic? - SELECTIVE CULTIVATION, isolation, and maintenance for pathogenic
A: FALSE (eukaryotic – unicellular) and non pathogenic organism especially YEAST, MOLD AND ACIDIC
BACTERIA
Q: Do they grow best at room temp? - primary culture medium used for the cultivation of fungi
A: TRUE - Dextrose - source of energy and carbon source
Q: Are they susceptible to antibacterial agents? - Agar - solidifying agent
A: FALSE - antifungal - Chloramphenicol and gentamicin - antibacterial
- the reason why antibiotics are not effective against fungi is because 3. Glucose, maltose, sucrose, fructose agars
fungi are eukaryotic so their genes and gene products mostly resemble that 4. India ink
of human genome already and antibiotics are made for prokaryotes and 5. Serum (medium for germ tube production)
simple structures. Fungi cell wall are more resistant that’s why we need
stronger medications for a longer period of time. OBSERVATION
Rhizopus CANDIDA ALBICANS
2. stolon Gross examination in SDA
Penicillium is a saprophytic fungus, and under the microscope naa siyay
3. sporagiophore - supports sporangium
characteristics
4.sporangiospore (asexual structure) Colonies with white to cream colored, smooth
5. sporangium - mitotic spore production globrous, and yeast-like appearance
For Aspergillus and Penicillum
What is a Penicillium (cause this a cell term used to describe a structure man sad) Microscopic Examination under Oil Immersion Objective

Whats a phialide
- Conidia prod by a “vase-shaped” conidiogenous cell
Metulae - outermost branches of a conidiophore from which flask-shaped
phialides radiate
Conidiophore - specialized hyphae that conidia can be formed from
Conidia
- Asexual reproductive structures (mitospores) prod either from the
transformation of vegetative yeast or hyphal cell or from a
specialized condiogenous cell w/c may be simple or complex and
elaborate

Reading: purple stain

Q: Does penicillium has chains of conidia?that are produced from Interpretation: Gram positive
phialides? Small and oval/spherical calls and may be budding; has elongated and
A: TRUE thread-like filaments, and presence of chains of elongated cells that
Q: do they have brushlike structure are constricted at septations
A: TRUE
GERM TUBE PRODUCTION using serum as a medium CRYPTOCOCCUS NEOFORMANS
Gross examination of Cryptococcus neoformans in SDA 3. What is the mode of transmission of Cryptococcus neoformans and its
- Colonies are white to cream similar to Candida albicans and mucoid major clinical manifestation?
(because of the capsule) - Mode of Transmission: contact with avian species specifically, pigeon
feces; inhalation of desiccated or dried yeast cells in pigeon excreta
- Major Clinical Manifestation: Cryptococcal meningitis with common
symptoms of:
• headache
• nausea
• vomiting
• mental changes, including confusion, hallucinations, and
personality changes
India Ink Preparation: C neoformans • lethargy
• sensitivity to light
bl k l i
What are the different morphologies of Candida albicans? How can you
differentiate one from the other?
Microscopic Examination of Cryptococcus neoformans The different morphologies of Candida albicans are yeast, hyphae,
stained with India Ink and pseudohyphae. These can be differentiated by examining their
morphologies under the microscope using the Oil Immersion
Objective wherein yeast cells are oval/ spherical and budding, true
hyphae are elongated and have thread-like filaments, and
pseudohyphae are chains of elongated cells that are pitched or
constricted at the septations between cells.

QUESTIONS
1. What are the different morphologies of Candida albicans? How can
No constrictions you differentiate one from the other?
Short hyphal filamentous extension arising laterally from a yeast cell The three morphologies of Candida albicans can be clearly distinguished
with no constriction at the point of origin under the Oil Immersion Objective .
Germ tube = represents the initial stage of true hyphae formation YEAST: unicellular organisms; oval-shaped; budding; can grow in 37⁰C
HYPHAE: multicellular elongated, thread-like structure; branching
Sugar Fermentation of CANDIDA ALBICANS filaments that make up the mycelium of a fungus; occurs in filamentous
fungi; has visible septa or is aseptate
Glucose: A/A Maltose: A/A Sucrose:NC Fructose: NC PSUDOHYPHAE: occurs in unicellular fungi; ellipsoid-shaped; connected
as a chain with constrictions at the site where septa is located
2. What are the two morphologic tests that distinguish Candida albicans
from other Candida species?
- Germ tube production in serum incubated at 37⁰C for 60-90 minutes
(or until 3 hours)
- Production of large spherical chlamydospores inoculated in a nutrient
deficient agar
What are the two morphologic tests that distinguish Candida albicans
from other Candida species?
A. Germ tube production
- in serum; incubated at 37 degrees Celsius at 60 - 90 minutes
B. Production of large spherical chlamydospores when inoculated in
nutritionally deficient media
What is the mode of transmission of Cryptococcus neoformans and its
major clinical manifestation?
contact with feces from avian species, specifically pigeon droppings
or from unwashed raw fruit. Its major clinical manifestation is chronic
meningitis, which can resemble a brain tumor, brain abscess, and etc.
Reservoir - pigeon excreta
Transmission - inhalation of basidiospores or dessicated or dried
yeast cells
Signs and symptoms - lung infection or pulmonary is the first sign &
symptom
If the body or human host cannot contain the infection in the site, C
neoformans can disseminate thru out the body but this organism is
neurotropic (predilection to migrate to CNS) so the major clinical
manifestation is MENINGITIS then it can turn to meningoencephalitis
1. Best therapy for cryptococcal infection; standard treatment of
cryptococcal meningitis
A: amphotericin b + flucytosine
2. Is Cryptococcus neoformans negative or positive with the india ink
A: Positive
India ink
- Creating a semi opaque background making the capsule of the yeast
visible
- is actually a type of negative staining wherein instead of staining the
microorg of interest, it is staining the background to give good
contrast between the background and the microorg that’s why we
were able to highlight the presence of the capsule of Cryptococcus
neoformans which is a chraracteristic feature of this microorganism.
And we can report it as INDIA INK POSITIVE
- Particles cannot penetrate the large polysaccharide capsule of C
neoformans that’s why it appears refractile (reason why C
neoformans is positive)
Simple staining - one stain is used
1. Positive staining - organism is stained
2. Negative staining - background is stained
Differential staining
Gram staining - Hucker’s method
Acid Fast staining
Germ tube production
- no constriction present (it is very specific for germ tube it should not
have a constriction at its base)
- Hyphae like extension in germ tube is a true hyphae because germ
tube represents the initial stage of true hyphae formation
Candida tropicalis
used as a control
Does not typically produce a germ tube but can produce a
pseudo-germ tube
blastoconidial germination with constriction
Pseudo-germ tubes - has a constriction at its base
Principle: formation of germ tube is associated with increased
synthesis of protein and ribonucleic acid; germ tube solution contains
tryptic soy broth and fetal bovine serum, essential nutrients for
protein synthesis; it is lyophilized for stability; germ tube is one of the
virulence factors of Candida albicans
Germ Tube Test
- presumptive and rapid test of Candida albicans
- Positive = short hyphal filamentous extension arising laterally from a
yeast cell, with no constriction at the point of origin; germ tube is hald
the width and 3/4 times the length of the yeast cell and there is no
presence of nucleus
- Negative = no hyphal filamentous extension arising from a yeast cell
or a short hyphal extension constricted at the point of origin
Candida dubliniensis is also positive
80% of spp positive for Germ Tube is C albicans
The rest is Candida dubliniensis
- Follow an incubation time strictly more or less 1-3 hours
- If it exceeds 3 horus, pseudohyphae forms which means that it will
become FALSE POSITIVE and might be misidentified as a germ tube
PSEUDOHYPHAE VS TRUE HYPHAE
- Pseudohyphae= produced by the failure of elongating budding cells to
separate, so that’s why they don’t have cell wall septations , (no
septum just constrictions); described as chains or links of sausages;
they have cell wall and has constrictions
REMEMBER: Psuedo = constriction
- Pseudo germ tube- constriction at its base
- True hyphae= if its truly divided then its true hyphae , like it has
septum
TEACHERS - ginotes nako para ma arrange
What are the causative agents for your systemic mycoses
A: (4)
1. Histoplasma capsulatum
2. Coccidioides immitis
3. Blastomyces dermatitidis
4. Paracoccidioides brasiliensis
1. The 2 opportunistic mycoses:
A: Candida albicans and Cryptococcus neoformans
Candida albicans
Normal flora of Candida albicans: found in mucosal membranes in the
body and GIT, GUT, URT
has a spectrum of disease - the immunocompetent (Candidiasis) and
immunocompromised
common disorder is Vulvovaginitis/ Vulvovaginal candidiasis
yeast infection in the vagina caused by strings of Candida
Clinical manifestation: watery, thick and white cottage cheese like
appearance (REMEMBER!)
Medical management: antifungal medications; -azole
Nystatin and Mycostatin (no -azole but still and antifungal)
Other diagnostic test for the determination of Candida albicans
(mentioned in the lab: gram staining, germ tube, sugar
fermentation, culture and sabouraud dextrose agar)
Corn Meal agar (we are able to appreciate the chlamydospore)
10 % Potassium Hydroxide (KOH) – example you have a skin
infection (skin scraping; you apply this to the specimen before I
examine it under the microscope; its an alkali), we use this; this is
the first test that we order if we want to visualize fungal
elements
(side notes: if bacteria gani, 1st test we order kay gram staining)
(1–3)-β-D-Glucan Assay - highly desirable; it provides a
noninvasive test method which is designed to diagnose invasive
fungal infections
DNA and PCR (polymerase chain reaction)
Serologic test
Cryptococcus neoformans
Meningitis
increased ICP (pressure inside the brain tissue or skull)
headache; nuccal rigidity
Nursing intervention: check vital signs especially BLOOD
PRESSURE (increased ICP = increased systolic blood pressure,
decreased diastolic
Nursing intervention: elevate the head of the bed of the patient
to at least 30 degrees
staining methods that we can use in the determination of
Cryptococcus neoformans
a. (india ink- stains the background)
b. Mucicarmine stain -stains the polysaccharide capsule of C.
neoformans
c. Cryptococcus neoformans Gram Stain
d. India ink preparation
usually positive with immunocompromised patients, such as those
with with hiv/aids
negative for non hiv or aids patients; india ink is usually positive at
only about 50% (50% of the time ra siya positive)
Situation: If it is negative and patient is suspected to have
Crypotococcal meningitis but is negative for HIV, India ink is ordered
because CSF is negative. What other test can I order to support my
diagnosis of Cryptococcal meningitis ( it’s a serologic assay)
A: CALAS - Cryptococcal Antigen Latex Agglutination System or Test
uses CSF
qualitative and semi-quantitative test system for the detection of capsular
polysaccharide antigens of Cryptococcus neoformans
both diagnostic and prognostic value since progressive disease is usually
accompanied by increasing antigen titers
CSF titers of 1:4 or less are presumptive evidence of central nervous system
infection by C. neoformans; additional follow-up and culture are strongly
recommended. CSF titers of 1:8 or greater from patients with meningitis
strongly suggest infection by C. neoformans.
Doctor usually orders the CALAS along with culture and sensitivity
*No question, gishare ra ni sir
- Candida albicans sputum specimen is not released, but if the patient
it from the ICU, it can be released; but if the specimen is sterile like
blood, Candida is leaked(?)
- Candida spp are normal flora of the specific parts of the body so if
they are isolated from the sterile sites of the body like CSF or blood, it
is automatically significany
- if we isolate it from sputum and urine, it can be due to contamination
especially if the patient is debilitated or immunocompromised or has
catheter
- Candidemia - presence of Candida in the blood; may be transient
because of the contamination
- IF WE ISOLATE FUNGI ESP CANDIDA AT STERILE SITES, IT IS ALREADY
SIGNIFICANT
What specific culture medium can we use to isolate C. neoformans, in
which it would grow brown to black on this agar
A: Niger seed agar - selective medium for the growth of Cryptococcus
neoformans, C gatii complex
What pigment is present is present is responsible sa brown to black
colonies, and what enzyme catalyzes the formation of that pigment?
A: pigment= melanin produced by phenol oxidase (enzyme)
What types of agar should be avoided in growth of C neoformans,
because the addition of this antifungal will inhibit C. neoformans
A: media with Cycloheximide!!
- inhibits Cryptococcus neoformans
- this is why we culture in plain SDA
- antifungal
- inhibits rapid growing fungi
- added when one of you suspect slow growing fungi (C neoformans)
Chloramphenicol - antibacterial agent
What makes Candida albicans different from nonpathogenic organisms:
germ tube test and the ability of the Candida albicans to grow
clamydospores in nutritionally deficient media
What makes Cryptococcus neoformans different from nonpathogenic What is the germ tube of Candida albicans?
organisms? exhibits true hyphae
A: the presence of phenol oxidase which eventually produces melanin and if a germ tube is already positive, we can release a preliminary result
their ability to grow in 37 degree Celsius of C albicans or C dubliniensis, however, we have to couple the
results of Germ tube production to that of VITEC
However there is another species that can actually grow at 37degree
fungal infections takes a longer time to treat
Celsius and also produce phenol oxidase what specie is that?
A: Cryptococcus gattii - similar to C neoformans phenotypically; difficult to
From patients infected with Candidiasis and Cryptococcosis
differentiate both
Candidiasis - collect specimens by oral swab, vaginal swab, biopsy of
C NEOFORMANS AND GATII specimen, skin swab, blood and urine
pathogenic species of Cryptococcus Cryptococcosis - sputum, bone marrow, blood, BIL
ability to grow in 37 degrees Celsius Specimens must be transported within 2 HOURS and is already plated
production of black case and presence of phenol oxidase in the appropriate agars within 2 hours to avoid mishandling of the
specimen and to avoid bacterial over growth
Glycine assimilation test
doubling time of the bacteria is shorter than fungi that’s why we have
To differentiate C gatii and neoformans
to plate our specimens right away ASAP
C neoformans/gatti (not sure kay lahi gi ingon sa lain sec pls send
help) is the only one who is able to use glycine
How do you prove that a yeast is dimorphic?
IF IT BOTH CANNOT BE DIFFERENTIATED: Cryptococcus
incubate the yeast or culture at 36 degrees Celsius
neoformans/gatii or Cryptococcus complex (how we report it in the
at 36 degrees Celsius, dimorphic fungi demonstrate yeast forms and
lab)
mycelial mold forms at 28 - 30 degrees Celsius
Initial infection: Primary pulmonary infection
primary pathogens are dimorphic
Route of entry: Inhalation of dry, minimally encapsulated, and easily
aerosolized yeast cells; dessicated yeast cells or smaller basidiospores
What do you call the specific type of dimorphism exhibited by
Environmental reservoir of spores and yeast cells: Bird (pigeon)
Histoplasma?
droppings/excreta
thermally dimorphic fungi - dependent on temperature
C. neoformans = neurotropic – meaning it has a predilection to infect
CNS
What kind of urease test is done?
What are the important parts of the history that you should be able to extract
RAPID UREASE TEST - positive within a few hours
when you suspect meningitis (because dili pwede C neoformans dayon)
1. Sexual history (multiple sexual partners; HIV patients)
Traditional urease test - some strains of rotten morula, candida, and
2. ORGAN TRANSPLANT - also immunocompromised and they take steroid to lower trichosporon becomes positive in this test
the immune system to allow the body to accept the foreign transplanted organ
3. history of chemotherapy
4. with leukemia and hematogenous disease cannot mount the effective immune
response for fungi
5. Medication history ( antibiotic and steroids)
- too much antibiotic, the normal flora of the body might be changed already so
there will be overgrowth of the normal microbiota fungi
When can we say that yeast cells are significant?
A: Disregard the yeast cells = sputum, mouth swabs, vaginal swabs
Important and yeast cells = CSF, blood, sometimes urine
Why was the serum used in the Germ tube test?
Serum has essential nutrients for protein synthesis wherein the germ
tube can grow in proteinaceous media
incubate at 37 degrees Celsius for 2-4 hours
in 60-90 minutes = appreciate germ tube production in Candida
albicans