EXERCISE 1 : CONTROL OF MICROBIAL GROWTH

A. PHYSICAL METHODS OF CONTROL HEAT

Thermal death point- lowest temperature where bacteria are killed
Thermal death time shortest time of bacteria to be killed
MOIST HEAT
Method
-

1. Autoclaving / steam
under pressure

Bacillus stearothermophilus
- detect effectivitiy of
autoclave
- strip turns black
-

2. Boiling
3. Fractional/
tyndalization/
interminent

Action
Coagulation of
protein
Culture media

Coagulation of
protein
Kills only vegetative
not spores
Dental instruments.
Feeding bottles
Coagulation of
protein
Culture media that
cant withstand
autoclave

Germination spores
transformed into vegetative
cell then destroyed
- Coagulation of
protein
- Used with high
protein media
(which cant stand
autoclaving)
- Dorset egg medium,
Loffler serum,
Lowenstein jensen
HTST high temp short
time

4. Inspissation

Apparatus

Standard Condition

Autoclave

121C 15- 20 mins 15 psi

100C 10-20 mins

boiler
*timed when it starts to boil

100C for 30 mins for 3

consecutive days with
incubation
Arnolds sterilizer

*to allow bacteria to

germinate and to be killed
the following day

75C - 80C 2 hrs for 3

consecutive days

Inspissator

72C 15 s
LTH- low tem holding
5. Pasteurization

pasteurizer

60-63C 30 mins

UHT- ultra high temp

72-140-72C 3s
For dairy milk, alcoholic
beverages
DRY HEAT oxidation of cellular constituents of bacteria; less effective compared to moist heat because oxidation is
slower than coagulation
Method
1. Hot air

Action
Oxidation
Glassware, petri dishes
Spore strip: green then black
Bacillus subtilis var. Niger

Apparatus

Standard Condition

Oven

160-180C for 1.5 2

hours

2. Open flame
3. Incineration
4. Cremation

Burning to ashes
Needles, loops, inoculating needle (red hot)
Burning to ashes
Waste products
Sputum cups, infected animals, wound
dressings
Cremate bodies with HIV

Bunsen
burner
Incinerator

870-980C

B. CHEMICAL METHODS OF CONTROL :

DISINFECTANT AND ANTISEPTICS
Sterilization process of killing or destroying
microorganisms and microbial spores
Fungi- fungicidal
Spores- sporicidal
Virus- virocidal

4. throroughly clean between fingers, under

fingernails and rings and up to the wrist for
atleast 15 seconds
5. rinse hands in downward position
6. dry with paper and hand towel
7. turn off faucet with the unused paper towel
to prevent contamination
EXERCISE 2: PREPARATION OF CULTURE MEDIA

Bacteriostatic- inhibits the growth of microorganisms

Bactericidal kills the growth of microorganisms
Disinfection inhibiting the growth of microorganisms
Disinfectants- chemical agent that kills bacteria :
vegetative cells only
Antimicrobial agent / drug chemical agent that kills /
inhibits without damaging the body tissue
Sanitizer- agent that limits growth of bacteria to a safe
level
Antiseptic- prevents growth of microorganisms by
inhibiting their growth / activity
ex. Lysol
Mechanism of action:
Lysol
-

Bacterial protein denaturation

Cytoplasmic membrane destruction
Inactivation of enzymes

Alcohol( 70% alcohol)

- Cell membrane destruction
- Lipid dissolution
- Protein denaturation
Soap - Disruption of cell membrane
Sepsis- presence of bacteria in a system
Asepsis- absence of bacteria in a system
Biological safety cabinet
- Protecting self and microorganisms you are
working on
- Protected by sterilization by UV light / passage
of filters
- Protected from aerosols
Filters:
HEPA high efficiency particulate air filter
ULPA ultra low particulate air filter
C. HAND SCRUBBING
1. wet hands with warm water
2. apply antimicrobial soap
3. rub to form lather, create friction and loosen
debris

CULTURE MEDIA
- Granular or powder form
- Material containing essential nutrients for growth
of bacteria
- Serves as food sand soil
Criteria
- Proper pH
- Sterile
- Free of inhibitory substance
- Adequate amount of water and salt
- Contain essential nutrients in proper
concentration
- Right moisture
Nutrients required:
1. Source of carbon
2. Source of nitrogen
3. Source of minerals and vitamins
4. Metabolic elements
Plated (500 mL Ernlenmeyer flask)
1. Weighing
2. Dissolving (hot plate)
3. Plugging (gauze)
4. Autoclave
5. dispensing
6. Formation (dispense in petri dish)
Tubed (beaker)
1. Weigh
2. Dissolve
3. Dispense
4. Plugging
5. Autoclave
6. Formation
Plated
- 2 plates/ student
- 20ml/ plate
20ml x 14= 280 = 300ml

Butt slant
- 6 loefflers/ group
- 10 ml/ tube
10ml x 6 = 60 = 100 ml

CAP- Chocolate Agar Plate

Slant
-

6 screwcap/ group
5ml/ tube

5ml x 6= 30 = 70 ml

Butt
-

1 wasserman/ student
3ml/tube

3ml x 7= 21 ml = 50 ml

Broth
- 3 wasserman/ group
- 2ml/ tube
2ml x 3= 6 = 10 ml

CLASSIFICATION OF CULTURE MEDIA

A. According to physical state / consistency
1. Liquid- no solidifying agent (ex. visible
growth of bacteria; nutrient broth becomes
turbid)
2. Solid- contains agar (1.5 3% solidifies by
using red algae polysaccharide)
3. Semi-solid- used for motility test, 0.5-1%
agar
Gelatin- diagnostic test
Agar- 38C solidifies, 100C liquefies
solidifying agents- albumin , agar, gelatin
B. According to function
1. Basal/simple/ordinary
Basic- support growth of non-fastidious
bacteria
NB, NA- general culture media
Composition of simple solid mediumpeptone, beef extract, water, agar
2. Enriched- used to support growth of
fastidious bacteria ( difficult to grow)
NA + enriching salts- blood, serum, ascetic
fluid
BAP- Blood Agar Plate

3. Differential- allows differentiation of 2 or

more bacteria; incorporated is indicator
Mannitol Salt Agar
- Phenol indicator
- Yellow halo colonies (Staphylococcus
aureus)
- Red / pink colonies (Staphylococcus
epidermis)
Mac Conkey Agar
- Indicator- neutral red
- Lactose fermenters: red, pink, purple
- Non- lactose fermenters: colorless
Simmon Citrate (source of carbon) Agar
- Indicator: bromthymol blue
- Original color to green
- When it becomes Prussian blue: sole
source of carbon
4. Selective- incorporated, not alter growth of
undesired microorganisms, with inhibiting
salts
Alcohol, chloral hydrate- prevents
swarming of proteins
K tellurite, Na azide- inhibits growth of
gram negative bacteria
Gentian violet, sodium desoxycholate,
bile salts- inhibits the growth of gram
positive bacteria
C. According to composition
1. Synthetic/ chemically defined/ complex
2. Non-synthetic/ chemically undefined
D. According to form
1. plated
2. tubed
EXERCISE 3: TRANSFER OF BACTERIA: ASEPTIC
TECHNIQUE
Microbial techniques- methods employed for study,
cultivation and growth of bacteria and other species
Inoculation- process of implanting/ transferring
microbes/ infectious materials into culture media
Materials: inoculating needle, inoculation loop,
different culture media
Culture- growth of microorganism on nutrient medium
Colony- visible growth bacteria of deposited on the
surface of solid media
Fishing- picking up of single colony for transferring into
different culture media or smear
TYPES OF CULTURE

1. Pure culture- contains one species of

microorganism
2. Mixed culture- contains 2 or more species of
microogranims
Ex. e.coli/ kleb
3. Contaminated culture accidentally contains
more 1 than species of microorganism
4. Stock pure culture of microorganisms used as
source of supply or for research

Roate 90, streak, incubate for 24 hrs, 37C

PRACTICAL:
Pure culture
1. Plated- radial simple
Tubed- butt, butt slant, slant, broth
2. 18- 24 hrs at 37C
Mixed culture- always start in plated medium

Radial- flame sterilize, streak

1.
2.
3.
4.
Simple

Plated medium (clock method)

Incubate for 18- 24 hrs at 37C
Get from 3rd quadrant
Plated- radial simple
Tubed- butt, butt slant, slant, broth

EXERCISE 4: CULTIVATION OF BACTERIA,

MICROBES IN THE ENVIRONMENT
Whole colony:
punctiform
circular
rhizoid
irregular

Clock method
Surface:
smooth, glistening
rough
wrinkled
dry, powdery
METHODS OF INOCULATING MICROORGANISMS IN
TUBED
1. Butt
- Fish out colony from plate using inoculating
needle
- Stabbing the butt portion (1/4)
2. Butt slant
- use of inoculating needle
- by stabbing and by streaking ( zigzag
motion)
3. Slant
- use of inoculating needle and loop because
there is no butt portion
- by streaking
4. Broth
- Rub side of the test tube until it becomes
turbid
5. Plated (clock method)
- Lines of streaking are parallel to each other
but should not overlap with other lines,
flame sterilize
- Rotate, restreak, touch last two lines of the
previous streak, cover more than 1/3 of the
surface, flame sterilize

Edge:
entire
undulate
lobate
curled
Elevation:
flat
raised
convex
pulvinate
umbonate
size:
small
pinpoint
pinhead
large
EXERCISE 5: STAINING METHODS:
A. SIMPLE STAIN
- Crystal violet, methylene blue, safranin,
malachite green
1. Drop of water / NSS + small amount of
growth using loop or needle
2. Spread, heat fix
3. Stain for 1-2 minutes
4. Wash tap water , blot dry

5. OIO

B. DIFFERENTIAL STAIN: GRAM STAIN

1.
2.
3.
4.

Smear
Crystal violet (1 min), wash water
Grams iodine (1 min), wash water
Decolorize with acetone- alcohol/ 95% ethyl
alcohol, wash water
5. Counterstain with safranin (30 s) wash water
6. Blot dry, OIO
Positive: Purple Negative: Red
C. DIFFERENTIAL STAIN: ACID FAST STAIN
1. Prepare and heat fix sputum
2. Carbol fuchsin
3. Water bath. Steam for 5 minutes, adding
more carbol fuchsin
4. Wash with dH2O, decolorize acid alcohol
(15-20s), Wash with dH2O
5. Counterstain methylene blue (1min)
6. Wash with dH2O,blot dry, OIO
Positive: Red Negative: Blue

Corynebacterium
diptheriae
Kleb- Loefflers bacillus
gram (+)
irregular bacilli with Babes
Ernst bodies ;club-shaped
w/ barbed ends ; X,Y,V or chinese characters
arrangement
Gram staining
Neisseria gonorrhoeae
gram (-)
cocci in pairs
gram staining

D. SELECTIVE STAINING: SPORE STAIN,

CAPSULE STAIN
Spore: Bacillus subtilis
Fulton Schaeffers method
dH2O drop, diluents
primary stain: malachite green (10 minutes then wash)
counter stain: safranin (1 minute)

Staphylococcus aureus
gram (+)
cocci in clusters
gram staining

Salmonella typhosa
gram (-)
short bacilli arrange singly
gram staining

Capsule: Klebsiella pneumonia India ink method

Drop of india ink (diluents) + inoculums then spread
No fixing

EXERCISE 5: COMPOUND MICROSCOPE:

FOCUSING
Pseudomonas
aeruginosa
gram (-)
short bacilli arranged
singly
gram staining

Diplococcus pneumoniae
gram (+)
cocci in pairs
gram staining

Vibrio cholera
Comma bacillus
gram (-)
curved bacilli, comma shaped
gram staining

Spirillum volutans
gram (-)
spiral shaped
gram staining

Escherichia coli
Colon bacillus
gram (-)
short bacilli arranged singly
gram staining

Bacillus subtilis
gram (+)
bacilli in chains
gram staining
Escherichia coli
gram (-)
bacilli in singles
Sarcina lutea
gram (+)
cocci in groups of 8
gram staining

Sensitivity testing
Mueller Hinton Agar
Conc 38 g / L x 200 mL

S. aureus specimen A
E.coli specimen B
-

Important in management of infectious diseases

particularly if susceptibility pattern of
microorganisms cannot be predicted

Manner of Reporting
Susceptible/ Sensitive growth is inhibited in vitro ,
effective against bacteria
Resistant- not effective, against growth of bacteria
Intermediate
Susceptibility test
1. Dilutions Broth /Agar dilution
2. Disk Diffusion Kirby Bauer Method
Broth Dilution
- Different concentration of chemotherapeutic
agents by serial dilution, uses 2 fold dilutions
- 1000 / 2 500 250 (conc. of antibiotic /
chemotherapeutic agent)
Observe macroscopically
1000 - turbid, not effective

Mycobacterium
tubercolosis
Tubercle bacilli / Kochs
bacillus
Acid fast
Slender bacilli in serpentetive
cord pattern
Ziehl Neelsen acid fast stain

Agar Dilution
- Uses petri dish
- Different concentration of chemotherapeutic
agents
- Heated agar (not solidify yet)

non- acid fast

cocci in tetrads, chains

allow to solidify
then streak,
incubate 18
-24 hrs,
observe
colonies

EXERCISE 6 MOTILITY OF BACTERIA

Consider:

Hanging Drop Preparation

- Examine microscopically
- Concavity slide/ depression slide
- Materials: Vaseline or white petroleum jelly (to
avoid dehydration), applicator slide
Brownian movement: bombardment of molecules of
water

Plating medium
depth of medium
- 4mm high
- Too thick false resistant
- Too thin false susceptible
Size of inoculum
- Compare sa 0.5 MacFarland (1.1575% Barium
Chloride Dihydrate + 1% Sulfuric Acid)
pH of medium
- 7.2 7.4

Motile: Bacillus subtilis

Non motile: Staphylococcus aureus
EXERCISE 7: ANTIBIOTIC SUSCEPTIBILITY
TESTING
ANTIBIOTIC SUSCEPTIBILITY TESTING
Plated medium (Erlenmeyer flask 200 mL)

Disk Diffusion
- Kirby Baurer
- MHA 1. Batch to batch uniformity 2. Low in
sulfonamide and tetracycline inhibitors

Streaking : overlapping using sterile cotton

swabs
Dont place 24 mm near center
Dont place 10-15mm periphery
If too thick, you need to dilute. Diluents
NSS/NB
Composition 0.5 MacFarland ( 1.1mL 75% BaCl2
99.5 mL 1% H2SO4)
Zone of inhibition measure diameter, more
susceptible , compare with reference
6mm measurement of antibiotic disk

EXERCISE 8 BACTERIA OF THE RESPIRATORY

TRACT, THROAT CULTURE
Blood agar plate 28 g / L
Mannitol salt agar plate 111 g / L
Chocolate agar plate 28 g/L
Phenylethyl alcohol agar 35.5 g/L
Eosin methylene blue 36 g / L
BAP
hemolytic: complete with
clear zone ( pathogenic
Pinhead, creamy white to
Staphyloccoci)
yellow, convex, smooth
glistening colonies
hemolytic no zone of
hemolysis ( nonpathogenic Staphyloccoci)
hemolytic: complete with
clear zone ( pathogenic
Staphyloccoci)
Pinpoint, flat gray
translucent colonies

Large, gray mucoid

colonies
Large swarming, spreading
colonies with mousy or
burnt chocolate odor

hemolytic incomplete
greenish zone of
hemolysis
hemolytic no zone of
hemolysis ( nonpathogenic Staphyloccoci)
With or without hemolysis
( gram enteric bacilli)
Proteus species (urine
culture only)

CAP
With greenish discoloration
( pathogenic
Pinhead, creamy white to
Staphyloccoci)
yellow colonies
w/out greenish zone ( nonpathogenic Staphyloccoci)
With greenish discoloration
( hemolytic Streptoccoci)
Pinpoint, flat, gray colonies

w/out greenish
discoloration ( ,
hemolytic Streptococci)

Large mucoid with or

Gram (-) enteric bacilli

without greenish
discoloration

PEA
pinhead
Catalase test
Mannitol
fermentation test
Coagulase slide
method
If (-), do coagulase
tube method
Make a smear

Pink violet colonies

Colorless colonies

pinpoint

Catalase
Make a smear

EMB
Lactose fermenting gram
(-) bacilli
Non lactose fermenting
gram (-) bacilli

Catalase test
- Colonies on slide
- 2 drops of 3% hydrogen peroxide
- (+) bubbles
PATHOGENECITY TEST FOR STAPHYLOCOCCI
A. Coagulase test
1. Slide method
- Human plasma + organism from BAP
- (+) clumping
2. Test tube
0.5 ml human plasma + organism from
BAP
- Incubate, clot 30 minutes
B. Mannitol Fermentation
Yellow colonies
Pink colonies

MSA
Mannitol fermenting
staphylococci species
Non- mannitol fermenting
staphylococci species

EXERCISE 9: BACTERIA OF UROGENITAL TRACT

(URINE CULTURE)

Pink violet colonies

Colorless colonies
-

MAC
Lactose fermenting gram
(-) bacilli
Non lactose fermenting
gram (-) bacilli

Multiply 1000 to get CFUs/ml

Gram (-) bacilli, singles