Microbiology Seminar 1

Topic No. 1 Speaker: Angelo Balcita, RMT Date: 10/07/2023 Transcribed by: AAA
*CALIBRATED LOOP/ INOCULATING LOOP
STAPHYLOCOCCUS AND STREPTOCOCCUS PT. 1

TABLE OF CONTENTS
1 Introduction
2 Staphylococcus

INTRODUCTION
*CULTURE MEDIA AGAR
- Take note of EXPIRATION DATE
 Beyond expiration date = may be penalized or
SUSPENSION

- Ex.
 BAP & MAC = urine
 Thioglycolate = wound
 BHI = blood
*STAINING AREA

*GLASSWARES

*BIOCHEMICAL TESTS

Citrate
SIM LIA TSI

Urease

*ANTIBIOTICS

- planted on MHA to detect if microorganism is resistant,

intermediate, or sensitive to antibiotics.

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 Microbiology Seminar 1
Topic No. 1 Speaker: Angelo Balcita, RMT Date: 10/07/2023 Transcribed by: AAA
*ASEPTIC TECHNIQUE pressure when the volume is constant
-process to sterilize inoculating loop/needle
 To avoid contamination Pressure ∝ Temperature

- When pressure is increased in a closed vessel, the

temperature increases proportionately
 i.e 15 pressure per square inch temperature
rises to 121 C

- This pressure and temperature is kept constant for 20

minutes during autoclaving

AUTOCLAVE PRINCIPLE
Pressure = 15 PSI
Temperature = 121 C
*OVEN/AUTOCLAVE/INCUBATOR Time = 20 minutes
- Oven = where glassware is placed after washing
- Autoclave = used to sterilize equipment and culture -sufficient to kill all vegetative forms and spores of the
media to avoid cross contamination organism
- Incubator = where planted/streaked medium are *PETRI DISH
stored - flat, shallow dish made of glass or
 Temp = 35 C plastic with a suitable lid

- used to culture different types of

cells, including bacteria and molds
 disposable
 check expiration date

Oven
Incubator
*MCFARLAND STANDARD (0.5)
- used to standardize the approximate number of bacteria
in a liquid suspension by comparing the turbidity of the
test.
 Standard
concentration = 0.5

- made up of
Autoclave  Barium cholride
 Sulfuric acid
STERILIZATION
- term referring to any process that removes or kills all -produces white precipitate = BARIUM SULFATE
forms of microbial organisms such as fungi, bacteria,
viruses, spore forms, etc. present on a surface CALIPER
contaminated in a fluid or in a compound such as - measure ZONE OF INHIBITION
biological culture media  measure if microorganism is resistant,
susceptible, or intermediate
- can be achieved by applying  Reaction of media (MHA) to antibiotic
 Heat
 Chemicals
 Irradiation
 High pressure
 Filtration
AUTOCLABE
- used to remove microorganisms and
spores using high pressure and high MICROSCOPE
temperature STEAM STERILIZATION A. PARTS OF A MICROSCOPE
- pressurized device designed to heat
aqueous solution above boiling point at
normal atmospheric pressure to
achieve sterilization

-ETYMOLOGY
 auto = self
 clavis = self-locking device
A. PRINCIPLE
- Boiling point of water is directly proportional to

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 Microbiology Seminar 1
Topic No. 1 Speaker: Angelo Balcita, RMT Date: 10/07/2023 Transcribed by: AAA

B. MAGNIFICATION OF LIGHT MICROSCOPE

Total Magnification = Eyepiece x

objective
Eyepiece Objective Overal
l
X4 (Scanner) X40
X10 (LPO) X100
X10
X40 (HPO) X400
X100 (OIO) X1000
BIOSAFETY CABINET
- used to protect personnel against biohazardous or
infectious agents and to help maintain quality control of
the material being worked with as it filters both the inflow
and exhaust air
A. TYPES OF BIOSAFETY CABINET
Class 1 personnel and environment
Class 2 personnel, environment & product
maximum protect for personnal,
Class 3
environment and product

B. WORKFLOW

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 Microbiology Seminar 1
Topic No. 1 Speaker: Angelo Balcita, RMT Date: 10/07/2023 Transcribed by: AAA
AGAR PLATE
BLOOD AGAR
MCCONKEY AGAR (MAC)
PLATE (BAP)

- detects GRAM (-) detects GRAM (+)

 McCONkey = Negative  BA(P) =
Positive
- FERMENTERS
a. Lactose = pink
b. Non lactose = yellow
c. Late lactose fermenters
- If both media have growth, collect from MCCONKEY
 NSS  Mcfarland  Culture & Sensitivity 
Biochemical testing
 Bacteria cannot be identified via culture alone,
proceed to BIOCHEMICAL TEST

GRAM (+) VS GRAM (-)

Gram (+) Gram (-)
Single-layered, Double-layered,
Cell wall
smooth (simple) wavy (complex)
Thickness 20 – 80nm (thick) 8-10nm (thin)
Peptidoglyca Thick layer/ multi- Thin layer/
n Layer layered single layered
Teichoic acid Present Absent
Lipopoly-
Absent Present
saccharide
Outer
Absent Mostly present
membrane
Lipid content Very low 20-30%
Resistance to
Susceptible Resistant
Antibiotic
Blue/Purple
Pink/Red (retain
(retain crystal
safranin)
violet/idone)
Color
- Red flag =
- (P)urple =
Negative ^_^
(P)ositive

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 Microbiology Seminar 1
Topic No. 1 Speaker: Angelo Balcita, RMT Date: 10/07/2023 Transcribed by: AAA
A. EXAMPLES OF BACTERIA - Acetic acid bacteria
- Lactic acid bacteria (yogurt)
GRAM (+) GRAM (-) (vinegar)
- Bidobacterium
- Xanthomas (xanthan
gum)
- Staphylocccus aureus
- Zygomonas (tequila)
- Mycobacterium tuberculosis
- Pantoea
- Anthrax
- Enterococci
- Escherichia coli
- Streptococcus pneumoniiiae
- Vibrio cholera
- Hemolytic bacteria
- Pseudomonas
- Clostrium botulinum
- Periodontal disease
bacteria
- all COCCI are gram (+) except = NVM
 Neisseria
 Vielonella
 Moraxella / Brahamella

- all BACILLI are gram (-) except = BL2AC2MEN

 Bacillus
 Lactobacillus
 Listeria
 Actinomyces
 Clostridium
 Mycobacterium
 Erysiphelothrix
 Nocardia
CULTURE & SENSITIVITY
organism is inhibited by the
serum concentration of the drug
that is achieved using the usual
Sensitive (S)
dosage
 Can be treated w/ the
drug
organisms are inhibited only by
the maximum recommended
dosage
Intermediate (I)  may be treatable with the
drug, but may require
adjusted dosage (YES
or NO)
resistant to the usually
achievable serum drug levels
Resistant (R)
 cannot be treated with
drug

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 Microbiology Seminar 1
Topic No. 1 Speaker: Angelo Balcita, RMT Date: 10/07/2023 Transcribed by: AAA
(+) Acid Fast Red
Non Acid Fast Blue Green

STAPHYLOCOCCUS
A. GENERAL CHARACTERISTICS
- characterized by
 Gram (+) cocci
 arranged in pairs, tetrads or
clusters
 non-motile
 non-spore formers
 facultative anaerobes
o can survive in both
Why some laboratory require to release GS first?
oxygenated and the deoxygenated
 To initially determine if bacteria is G (+) or (-) to
environment
provide appropriate antibiotics prior to final
reporting  Catalase (+)
o Reagent: H2O2
 To determine the type of microorganism (i.e.
bacteria or fungi) o (+) = bubble formation
GRAM STAINING  Oxidase (-)
“Come In And Stain”  Grows in 7.5 – 10% NaCl
- color B. CLINICALLY SIGNIFICANT SPP.
Crystal Violet 1 Staphylococcus aureus
1  Gram (+) = purple
(Primary stain) 2 Staphylococcus epidermidis
 Gram (-) = purple
Makes dye less soluble and 3 Staphylococcus saprophyticus
Iodine makes it adhere to the cell wall 4 Staphylococcus lugdunensis
2 5 Staphylococcus haemolyticus
(Mordant)  Gram (+) = purple
 Gram (-) = purple C. WHAT PUT PEOPLE AT RISK FOR SERIOUS STAPH
Washes away stain from gram INFECTIONS
95% Ethyl (-) cell wall
3 Alcohol -
 Gram (+) = purple
(Decolorizer)
 Gram (-) = colorless
Allows dye adherence to gram
Safranin (-) cell walls.
4
(counterstain)  Gram (+) = purple
 Gram (-) = red

NOSOCOMIAL INFECTION = hospital-acquired infection

D. VIRULENCE FACTOR
FACTOR EFFECT/FUNCTION
Helping attachment to host
Cell surface protein
tissues
ACID FAST STAINING (AFB) Polysaccharide Resst phagocytosiis
microcapsule
Protein A* Limits host immune response
“A- aureus”
Forming porins in cell membrane
Panton-Valentine
of host cells, resulting in cell
Leukocidin (PVL)*
death by leakage of its contents
a-hemolysin Red blood cells lysis
Chemotaxis Inhibiits chemotaxis of
inhibitory protein neutrophils and monocytes
Plays a role in adherence and
Extracellular
invasiion of host tissues, and also
adherence protein
has immune-modularity activity
Proteases, lipases, Enzymes contribute to host tissue
ZIEHL-
KINYOUN’S nucleases, destruction that facilitate the
NEELSEN
hyaluronatelyase, invasion and penetration of the
Method Steam (Heat)
Cold pholipase C, bacterium into host tissues
(Mordant) “Hoy si Neel”
elastase,
Primary Stain Carbolfuschin (red) staphylokinase
Decolorizer Acid Alcohol Toxic shock Causes toxic shock syndrome,
Malachite syndrome toxin-1 particularly in menstrual women
Counter stain Methylene Blue
Green (TSST-1)

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 Microbiology Seminar 1
Topic No. 1 Speaker: Angelo Balcita, RMT Date: 10/07/2023 Transcribed by: AAA
Causes scalded skin syndrome, 4. TOXIC EPIDERMAL NECROLYSIS
Exofoliative toxin
particularly in infants
Several types causes food
poisoning
Enterotoxins
 SEA, SEB, SED, SEG,
SEH, SEI
Clumping factors Clots blood plasma

STAPHYLOCOCCUS EPIDERMIS
A. VIRULENCE FACTORS
1. EXOPOLYSACCHARIDE “SLIME” OR BIOFILM
- enhances organism adhesion to implanted medical
devices and provides refractoriness to host defense
 Causes PROSTHETIC VALVE ENDOCARDITIS
B. INFECTIONS AND ASSOCIATED DISEASE
1 Hospital Acquired UTI
Prosthetic valve endocarditis (PVE)
STAPHYLOCOCCUS AUREUS 2
 (P)VE = EPIdermidis
A. VIRULENCE FACTORS
1. ENTEROTOXINS STAPHYLOCOCCUS SAPROPHYTICUS
- Heat stable exotoxins A. VIRULENCE FACTORS
 A-E, G-J - adheres to epithelial cells lining the urogenital tract
2. TOXIC SHOCK SYNDROME TOXIN -1 B. INFECTIONS AND ASSOCIATED DISEASE
- Formerly known as ENTEROTOXIN F - causes UTI in sexually active young females and in
(SUPEANTIGENS) older women with INDWELLING CATHETERS

- Causes menstruating-associated TSS STAPHYLOCOCCUS LUGDUNENSIS &

3. EXFOLIATIVE TOXIN HAEMOLYTICUS
- epidermolytic A. VIRULENCE FACTORS
toxin Gene mecA S. Lugdunensiis
Vancomycin resistance S. Haemolyticus
- Causes SSS & B. INFECTIONS AND ASSOCIATED DISEASE
Bullous impetigo
- endocarditis and UTI
4. CYTOLYTIC TOXINS
- a,b, y toxins
HEMOLYSIS PATTERN
5. ENZYMES
S. aureus b-hemolysis
- includes
Coagulase Negative
 Coagulase (Staphylocoagulase) Staphylococcus (CONS)
y-hemolysis
 Fibrinolysin (Staphylokinase)
 Protease
 Hyalurinase
 Lipase
 Beta lactamse (Penicillinase)

6. PROTEIN (A) = Aureus

- binds in the FC portion of IgG
A. INFECTION & ASSOIATED DISEASES
1. SKIN AND WOUND INFECTIONS (FFC)
- FFC
 Folliculits = smallest
 Furuncles
 Carbuncles
REVIEW QUESTIONS
1. Recommended temperature of autoclave
 121 C, 15 PSI for 20 minutes

2. What is the eyepiece magnification of LPO?

 X10

3. What is the eyepiece magnification of HPO?

2. SKIN SCALDED SYNDROME (SSS) – EXFOLIATIVE  X40
DERMATITIS
3. TOXIC SHOCK SYNDROME 4. What is the total magnification of OIO?
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 Microbiology Seminar 1
Topic No. 1 Speaker: Angelo Balcita, RMT Date: 10/07/2023 Transcribed by: AAA
 X1000

5. Biosafety Cabinet
- Vertical laminar airflow
- protects MT, Environment, and product
- air velocity = 75-100 linear ft/min
- recommended for hospitals
 Biosafety Cabinet 2

6. After gram staining, what will be the color of gram (-)

bacilli after decolorizer?
 Colorless

7. Staphylococcus
 Gram (+) cocci are arranged in pairs, tetrads, or
clusters
 Non-motile
 Non-spore former
 Facultative anaerobes
 Catalase (+), Oxidase (-)
 Grows in 7.5-10% NaCl

8. Exfoliative dermatitis that occurs primarily in newborns

& young children are caused by what bacteria
 S. aureus

9. What bacteria causes hospital acquired UTI and

Prosthetic valve endocarditis
 S. epidermidis

9.1 Causes UTI in sexually active young females and in

older women with indwelling catheters
 S. saprophyticus

10. Staphylococcus spp. exhibit what hemolysis

 B-hemolysis

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