MICROBIOLOGY

Pre-Lab discussions, Bailey, Mahon, Sir Marc’s discussion

Cultures ❖ Where the selection of the medium for

inoculation is based.
❖ Growth of microorganisms in a culture
❖ Most bacterial require no more than the
medium
ordinary culture media but a few needs
❖ Gold standard for disease identification
special media for growth or for aiding in
Types of Culture: their identification where cultural
characteristics and specific reactions may
1. Pure Culture be determinative factors for
❖ Only one species identification.
❖ Separates the pathogen from the normal ** if several plates will be inoculated for
microbiota a given specimen, arrange starting the
2. Mixed Culture most enriched to the most selective
❖ More than one species
❖ Examples are: BAP- S. aureus, S. Classification of Culture Media
epidermidis/ Staph and Srep
Physical state a. Solid Media
3. Stock Culture
b. Semi- Solid Media
❖ Several species contained in a separate c. Liquid Media
culture medium (1 species/culture Classification a. Synthetic Media
medium) b. Non- Synthetic Media
❖ Gron in a large volume of broth -> divide c. Living Tissue Media
into small freezer vials (to lengthen the Use a. Simple Media
shelf life of specimens at least a year) b. Enriched Media
❖ Used for academic and industrial Differential Media
purposes (storage) 1. Differential
Mildly Selective
Culture Medium Media
2. Differential
❖ Solid, liquid, or semi-solid substance
Moderately
designed to support the growth and
Selective Media
physiologic requirements of c. High Selective Media
microorganisms. d. Transport Media
❖ Composed of a mixture of nutrients such e. Enrichment Media
as carbon, nitrogen, sulfur, phosphorus,
hydrogen, oxygen, and buffers.
❖ Inhibitory agents (dyes, salts, and Further/specific Classifications:
antimicrobials) are added to facilitate the A. Physical state
isolation of the desired organism while 1. Solid- 2-3% agar, TSI, BAP, MAC
suppressing the growth of other 2. Semi- Solid- 0.5-1% agar, SI(T),
organisms present in the sample. decarboxylase media
Type of specimen 3. Liquid- no agar
B. Use » Used for organisms while in
1. Enrichment Media transport
» Selenite F- for S. typhi 7. Maintenance
» Proliferation of microorganisms » Viability of certain
before culture microorganisms while in storage
» Alkaline peptone water (APW)= » Brain Heart Infusion Broth
media of choice for Vibrio spp, (BHIB), NA slant
tetrathionate broth (gram – 8. Special
bacilli= Salmonella spp.) » Specific type of organisms
2. General Isolation/ Supportive/ » Lowenstein-Jensen media (M.
Simple/ Basal Media tuberculosis); cauliflower
» NA, TSB colonies, Thiosulfate citrate bile
» Routine use without added salts sucrose agar (Vibrio spp.)
supplement 9. Anaerobic
3. Enriched/ Nonselective Media » Not requiring oxygen because
» BA, CA- for Gonorrhea anaerobic
» Basic with supplement, special » Cooked meat media,
treatment, for fastidious thioglycolate broth
organisms 10. Susceptibility Testing
4. Selective » Mueller- Hinton Agar (MHA)
» SSA, MAC, BSA, XLD, MSA ▪ Depth: 4 mm
» Grow a specific organism only in ▪ pH: 7.2 – 7.4
the usage of antibiotics 11. Biochemical Testing
» Inhibitory substances » Based on biochemical reactions
(Inhibit gram +) » TSI
▪ Crystal/ gentian violet ▪ Slant- ferment lactose or
▪ Basic fuchsin sucrose
▪ Bile salts ▪ Butt- ferment glucose
(Inhibit gram -) ▪ Yellow (A)- ferments
▪ Potassium teliorite ▪ Red (K)- not ferments
▪ Sodium azide ▪ Gas (+)- cracks/ bubbles
(Inhibit swarming Proteus) ▪ H2S- blackening of
▪ Alcohol medium
▪ Chloral hydrate C. Composition
5. Differential 1. Synthetic/ Chemically- defined
» BAP, McC, ferments lactose (pink » Exact composition is known
colonies) » NA, MAC
▪ Rapid lactose fermenters 2. Non- Synthetic/ Complex
• Escherichia, » At least 1 component is not
Klebsiella, chemically defined
Enterobacter » Media with blood, serum, plant/
6. Transport animal/ yeast extract
» Stuart’s (all around media), Cary- 3. Living State/ Tissue Culture
Blair medium (for stool specimen » Living cells
 Chlamydia rickettsia and viruses,
» 3. Spread- plate- uncommon (petri dish and
Mycobacterium leprae (leprosy/
hansen’s disease cultured at
footpad of armadillo)
» McCoy’s cells (media of choice
for Chlamydia), Vero cells, HELA,
Hep2 cells
D. Dispensing/ Distribution
1. Plated (weigh- dissolve- autoclave-
dispense)
» Single layer
2. Tubed (weigh- dissolve- dispense-
autoclave)
» Broth, butt (deep), slant, butt
and slant (TSI)
Food Elements
» Type of nutrients needed by the
bacteria to grow
1. Peptone- protein source
2. Carbohydrate
3. Minerals- phosphates, salts,
potassium, magnesium
4. Meat/ beef extract- source
of amino acids, peptides,
proteins
5. Yeast extract- source of B
vitamins, Brewer’s yeast
(alcohol fermenter-
Saccharomyces cerevisiae)
6. Bile and bile salts- inhibits
growth of gram + bacteria
7. Growth factors- vitamins
and enzymes
8. Agar/ agarose-
polysaccharide gel- agarose
is purified from agar,
seaweed extract from red
algae, from Gelidium spp.
And Gracilaria spp.
Techniques:
1. Pour- plate- gold standard (quantitative)
2. Streak- plate- widely used in laboratory
agar)
Autoclaving- 121C, 15-20 mins, 15 lbs
Incubation Conditions:
1. Aerobes: 21% O2 + 0.03% CO2
2. Anaerobes: 0% O2, 5%-10% CO2 + 5%-
10% H2 + 80-90% N2 (anaerobe jars, bags
or chambers)
3. Capnophiles: 5%-10% CO2 + 15% O2
(CO2 incubator or bag; 3% CO2 in candle
jars)
4. Microaerophile: 5%-10% O2 + 8%-10%
CO2
**most bacterial, ideal incubation temperature-
35C
**aerobic cultures- examined after 18-24 hours
incubation
**anaerobic cultures- require 48 hours
incubation
STEPS IN MEDIA PREPARATION:
CALCULATIONS
1. How much powder is needed to make 10
plates of MAC?
Solution:
Total volume (TC) = # of plates x volume
of medium per plate
TV= 10 plates x 15 mL/ plate
TV= 150 mL
2. Determine the amount of medium (g) to
be weighed. Use the ratio of culture
medium powder to diluent specified by
the manufacturer
Solution:
MAC: 49. 53g
49.53g/1000mL= g/150mL
(49.53g x 150 mL=10000mL x X)/ 1000mL
X= 7.4295
WEIGHING STORAGE

- Graduated cylinder and weighing - Refrigeration= 2-4 degrees C

scale o Plates: upright
o Tubes: upright
DISSOLVING
**avoid freezing because ice crystals can damage
- Stirring rod/ automatic mixer
the media
- Broth; no heating
- Solid and semi-solid; >50 degrees C PERFORMANCE CHECK
- Use titration for pH
- Culture to a known pure bacterium
- Heat sensitive; do filtration
and observe growth characteristics
DISPENSING/ DISTRIBUTION - To check whether media is expired or
not
- Critical/ non-critical (estimated)
- Plugging of tubes CULTURE MEDIA SELECTION
- **tubed media are dispensed before
- Dependent on the type of specimen
they are autoclaved, plate media are
that you receive and the organisms
autoclaved before dispensing
most likely involved in the disease
AUTOCLAVING (appropriation)
o Example: stool culture: gram
- Temperature; 121 degrees C
-: C. difficile: Columbia
- Pressure; 15 psi
Colistin-Nalidixic Acid Agar
- Duration; 15 minutes

**loosen caps of tubes before autoclaving

STERILITY CHECK

- One random tube/plate per batch;

with growth after incubation in 35-
37 degrees C for 24 hours=
CONTAMINATED
o Plates: inverted
o Tubes: upright

Media Purpose Expected Results

MacConkey Agar Selective and differential A pink colony indicates lactose
medium for isolation of gram- fermentation while a translucent
negative bacilli such as colony indicated non-lactose
Enterobacteriaceae and fermentation
Pseudomonaceae
Blood Agar Differential and Enriched ❖ Alpha hemolysis/ incomplete
Medium that supports the hemolysis: colonies
growth of fastidious bacteria and
 inhibits the growth of some
bacteria like Neisseria and
Haemophilus. It supports growth
of Streptococci
Chocolate Agar Enriched medium for isolation of
most fastidious bacteria such as
Haemophilus and Neisseria spp.
Mueller Hinton Plain Agar Best medium for routine
antimicrobial susceptibility
testing using the Kirby- Bauer
diffusion method for non-
fastidious bacteria
Nutrient Agar General purpose medium for the
growth of a wide variety of non-
fastidious microorganism
surrounded by a green, opaque
zone (S. pneumoniae)
❖ Beta hemolysis/ complete
hemolysis: clear zone
surrounding the colonies (S.
aureus, S. pyogenes)
❖ Gamma hemolysis/ no
hemolysis (S. epidermidis)
❖ N. gonorrhea: small, gray to
white, mucoid colonies,
smooth consistency, destined
margins
❖ N. meningitidis: larger bluish-
gray, mucoid, round, convex,
smooth, moist, glistening
colonies
❖ H. influenzae: small, colorless,
moist colonies with
characteristic seminal odor
Zone of inhibitions (ZOI) around the
antibiotics
❖ E. coli: grayish to white colored
large, circular, convex colonies,
smooth and rough colonies
❖ S. typhi: smooth colorless, with
a diameter range of 2-4mm
❖ S. aureus: golden yellow
colored circular, convex and
smooth colonies (2-4mm),
opaque colonies

OBTAINING PURE CULTURE General Rule:

Principle: Rapid qualitative method for isolation,
dilution technique
❖ Essential for microbiological studies
❖ Attained so that a single cell occupies an
isolated portion of the agar surface. The
single cell will go through repeated
multiplication to produce a visible colony
of similar cells
❖ Usage of a nutrient agar to obtain pure
culture
Organisms that grow on BAP also grow on CA but
not all organisms that grow on CA will grow on
BAP
CA- supports highly fastidious organisms such as
Haemophilus and Neisseria gonorrhea
MAC- supports most gram – rods specially the
Enterobacteriaceae, differentiates lactose vs.
non-lactose fermenters
BA- where true hemolysis caused by respiration) (Actinomyces spp.,
microorganisms is found Clostridium spp., C. tetani, C. perfringens)
• Only organisms viable in the original
(clear) B-hemolysis- complete lysis
sample are able to be grown
(green) A-hemolysis- partial lysis

Streak Plate method

GRAM STAINING
❖ Most commonly used for isolation of
mixed culture of bacterial species. Gram stain
❖ Techniques include the simple, radial, ❖ Differential stain
and multiple interrupted (most ideal- ❖ Differentiates gram + and gram –
enables the microbiologists to have microorganisms
isolated colonies) ❖ Hans Christian Gram (1864)
Methods: Reagents:
1. T-streak- zigzag, 2-3x 1. Crystal violet- primary stain
2. Quadrant- divided by four quadrants, 1st 2. Lugol’s iodine- mordant
quadrant- complete growth, 4th 3. Acetone/ Ethyl Alcohol- decolorizer
quadrant- very important, where 4. Safranin O- secondary stain
isolation is obtained, 1x touch 3rd
quadrant only Gram +; teichoic acid + MgRNA + CV + iodine
3. Discontinuous- parallel streak complex -> insoluble Alcohol (blue)- thick murin/
peptidoglycan layer
Labeling of Plate:
Gram -; ^ lipids -> soluble in alcohol (red)
• Initials/specimen used/group
number/date performed (e.g., PROCEDURE:
ACYG/Ear/GroupA16/07-27-2021)
Gram + Gram -
1. Flood Blue Blue
with CV
SUBCULTURE PLATING Rinse
2. Flood Blue (CV- I Blue
Streak Plate Method with complex)
Iodine
❖ Usage of the sample obtained from pure Rinse
culture (primary plate/ NA) 3. Flood Blue Colorless
❖ Gather sample from isolated colonies with
and inoculate on prepared BA, CA, and alcohol
McC Rinse
4. Flood Blue Red
Limitations: with
• Will not grow obligate anaerobes safranin
O
(absence of oxygen to growth, uses
Rinse
sulfate, nitrate, iron, manganese for
QC: S. aureus (gram +), E. coli (gram -)
General Rules in Gram Stain results:
❖ all cocci are gram positive except-
Neisseria, Veilonella, and Moraxella
(Bramhamella)
❖ all bacilli are gram negative except-
[MCCBELLANGAWBEP]
o Mycobacterium,
Corynebacterium, Clostridium,
Bacillus, Listeria, Erysipelothrix,
Lactobacillus, Actinomyces,
Eubacterium,
Propionibacterium, Bifido-
bacterium, Nocardia
❖ All spirochetes are gram negative [BLT]
o Borellia, Leptospira (question-
mark appearance/ hooked
ends), Treponema
REASONS WHY (+) BECOMES (-)
- Removal of MgRNA by precipitation with
bile salts
- Autolysis, aging, and temperature of
incubation results to loss of cell wall
integrity
- Using an acidic solution of gram’s iodine
- Overdecolorization
REASONS WHY (-) BECOMES (+)
- Incomplete decolorization
- Thick smear preparation
ACID FAST STAINING
Koch’s bacillus (M. tuberculosis)
M. tuberculosis
- bundle of faggot
- dark pink/ red against a blue background
SPECIMEN COLLECTION
-at least 3-5mL of deep cough sputum
Steps:
Deep breath for 3x, 3rd time with cough to push
sputum out to be collected or inhalation of
saline/aerosol
TIMING:
-early in the morning
MORNING SPECIMEN- concentrated; preferred
2 samples for diagnosis
1 for antibiotic therapy
SMEAR PREPARATION:
1. Sterilize and label
2. Serial number and specimen number
3. Oval, continuous spiral motion, 3cm long x 2cm
wide
4. Airdry
5. Assess under printed paper with the text still
seen
6. Heat fix (pass by flame 2-3 times)
KINYOUN (COLD): Carbol Fuchsin (pink) ->
Tergitol /Phenol -> Acid Alcohol (3%)
concentrated HCl in 95% ethanol (faint pink) ->
rinse with H2O -> methylene blue (2-3 secs.) ->
rinse with H2O, blot on paper excess water ->
microscopic examination
ZIEHL NEELSEN/ EHRLICH’S (HOT): Carbol Fuchsin
(pink) -> heat (until steam is visible -> rest 5 mins.
-> Acid Alcohol (3%) concentrated HCl in 95%
ethanol (faint pink) -> rinse with H2O ->
methylene blue (2-3 secs.) -> rinse with H2O, blot
on paper excess water -> microscopic MBC (Minimum bactericidal concentration)
examination
Methods:
**cleanse every after staining and mordant
1. Broth Dilution Method
Acid Fast- Red/ dark pink ❖ Reference method
❖ Quantitative result
Non- Acid Fast- Blue
2. Agar Dilution Method
Reporting: 3. Agar Diffusion Test
a. Disc Diffusion Test (Kirby Bauer)
0- 0/300 OIF » Not widely performed
+n- 1-9/ 100 OIF Requirements: BBAASSSTI
1+- 10- 99/ 100 OIF 1. Bacterial 1 x 10 ^8
2+- 1-10/ 50 OIF inoculum size
2. Bacterial Mueller Hinton Agar
3+- >10/20 OIF medium
3. Antibiotic disc6 mm diameter
(refrigerated)
4. Atmosphere Aerobic, no CO2
ANTIBIOTIC SUSCEPTIBILITY TESTING :
5. Standard depth 4 mm (25-30 mL = 90-100
Kirby Bauer Disc Diffusion Method mm diameter
6. Standard pH 7.2 – 7.4
❖ Determines susceptibility of a bacterial 7. Standard 0.5 MacFarland (0.5 Barium
strain to a specific antibiotic suspension suspension 99. 5 mL
❖ Used to determine the most appropriate H2SO4)
treatment for the infection 8. Temperature 35 – 37 C
❖ Method of choice 9. Incubation 16 – 18 hours
❖ Determined after 24 hours of incubation time
❖ Streaking is free, no structure/ rules only
make sure the whole plate is streaked
Note:
and a ring is made against the wall of the
plate after streaking **No 2 discs closer than 24 mm
PROCEDURE: Antibiotics:
1. Make suspension NSS + isolated colony = 1. Broad spectrum- gram + and gram – (ex.
standard Tetracycline)
2. Get sample from suspension 2. Narrow spectrum- gram + or gram – (ex.
3. Streak in MHIA Penicillin (gram +))
4. Stand the plate for 3-5 mins.
Factors affecting the results:
5. Put the antibiotic discs (apply within 15
mins) 1. Suitability and pH of medium
6. Stand for another 15 mins 2. Amount of inoculums
7. Incubate 3. Thickness of the agar
4. Diffusion of the antibiotic discs
MIC (Minimum inhibitory concentration)
5. Incubation time
6. Incubation temperature

• Mueller Hinton Agar (MHA)

❖ Standard susceptibility medium for non-
fastidious bacteria
❖ Recommended medium because of
reduced concentration of inhibitors