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 Action Apparatus Standard Condition

1. Autoclaving / steam under pressure

- Coagulation of protein

- Culture media

Bacillus stearothermophilus- detect effectivitiy of

autoclave- strip turns black

Autoclave 121°C 15- 20 mins 15 psi

2. Boiling

- Coagulation of protein

- Kills only vegetative not spores

- Dental instruments. Feeding bottles

boiler100°C 10-20 mins

*timed when it starts to boil

3. Fractional/ tyndalization/ interminent

- Coagulation of protein

- Culture media that can’t withstand autoclave

Germination – spores transformed into vegetative cell then destroyed

Arnold’s sterilizer

100°C for 30 mins for 3 consecutive days with

incubation

*to allow bacteria to germinate and to be killed

the following day

4. Inspissation

- Coagulation of protein

- Used with high protein media (which can’t stand autoclaving)

- Dorset egg medium, Loffler serum, Lowenstein jensen

Inspissator75°C - 80°C 2 hrs for 3

consecutive days

5. Pasteurization

HTST – high temp short time

LTH- low tem holding

UHT- ultra high temp

For dairy milk, alcoholic beverages

pasteurizer

72°C 15 s

60-63°C 30 mins

72-140-72°C 3s

DRY HEAT – oxidation of cellular constituents of bacteria; less effective compared to moist heat because oxidation is slower than coagulation

Method Action Apparatus Standard Condition1. Hot air - Oxidation

- Glassware, petri dishes- Spore strip: green then black

Oven 160-180°C for 1.5 – 2 hours

- Bacillus subtilis var. Niger

2. Open flame- Burning to ashes- Needles, loops, inoculating needle (red hot)

Bunsen burner

3. Incenaration

- Burning to ashes- Waste products- Sputum cups, infected animals, wound

dressings

Incinerator

4. Cremation - Cremate bodies with HIV

B. CHEMICAL METHODS OF CONTROL : DISINFECTANT AND ANTISEPTICS

Sterilization – process of killing or destroying microorganisms and microbial spores Fungi- fungicidalSpores- sporicidalVirus- virocidal

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 filterULPA ultra low particulate air filter

C. HAND SCRUBBING1. wet hands with warm water2. apply antimicrobial soap3. rub to form lather, create friction and loosen

debris

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

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 carbon2. Source of nitrogen3. Source of minerals and vitamins4. Metabolic elements

Plated (500 mL Ernlenmeyer flask)1. Weighing2. Dissolving (hot plate)3. Plugging (gauze)4. Autoclave5. dispensing6. Formation (dispense in petri dish)

Tubed (beaker)1. Weigh2. Dissolve3. Dispense4. Plugging5. Autoclave6. 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

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 / consistency1. 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 testAgar- 38°C solidifies, 100°C liquefies

solidifying agents- albumin , agar, gelatin

B. According to function1. Basal/simple/ordinary

Basic- support growth of non-fastidious bacteriaNB, NA- general culture mediaComposition of simple solid medium- peptone, 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 PlateCAP- Chocolate 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 proteinsK tellurite, Na azide- inhibits growth of gram negative bacteriaGentian violet, sodium desoxycholate, bile salts- inhibits the growth of gram positive bacteria

C. According to composition1. Synthetic/ chemically defined/ complex2. Non-synthetic/ chemically undefined

D. According to form1. plated2. 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 mediumColony- visible growth bacteria of deposited on the surface of solid mediaFishing- 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 microogranimsEx. 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

Radial- flame sterilize, streak

Simple

Clock method

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

PRACTICAL:

Pure culture

1. Plated- radial simpleTubed- butt, butt slant, slant, broth

2. 18- 24 hrs at 37°C

Mixed culture- always start in plated medium

1. Plated medium (clock method)2. Incubate for 18- 24 hrs at 37°C3. Get from 3rd quadrant4. Plated- radial simple

Tubed- butt, butt slant, slant, broth

EXERCISE 4: CULTIVATION OF BACTERIA, MICROBES IN THE ENVIRONMENT

Whole colony:punctiformcircularrhizoidirregular

Surface:smooth, glisteningroughwrinkleddry, powdery

Edge:entireundulatelobatecurled

Elevation:flatraisedconvexpulvinateumbonate

size: small pinpointpinheadlarge

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 dry5. OIO

B. DIFFERENTIAL STAIN: GRAM STAIN1. Smear2. Crystal violet (1 min), wash water3. Gram’s iodine (1 min), wash water4. Decolorize with acetone- alcohol/ 95% ethyl

alcohol, wash water5. Counterstain with safranin (30 s) wash water6. Blot dry, OIOPositive: Purple Negative: Red

C. DIFFERENTIAL STAIN: ACID FAST STAIN1. Prepare and heat fix sputum2. Carbol fuchsin3. Water bath. Steam for 5 minutes, adding

more carbolfuchsin4. Wash with dH2O, decolorize acid alcohol

(15-20s), Wash with dH2O5. Counterstain methylene blue (1min)6. Wash with dH2O,blot dry, OIOPositive: Red Negative: Blue

D. SELECTIVE STAINING: SPORE STAIN, CAPSULE STAIN

Spore: Bacillus subtilis Fulton Schaeffer’s methoddH2O – drop, diluents primary stain: malachite green (10 minutes then wash)counter stain: safranin (1 minute)

Capsule: Klebsiella pneumonia – India ink method Drop of india ink (diluents) + inoculums then spreadNo fixing

EXERCISE 5: COMPOUND MICROSCOPE: FOCUSING

Pseudomonas aeruginosa gram (-)short bacilli arranged singlygram staining

Corynebacterium diptheriaeKleb- Loefflers bacillus

gram (+)irregular bacilli with Babes Ernst bodies ;club-shaped w/ barbed ends ; X,Y,V or chinese characters arrangementGram staining

Neisseria gonorrhoeaegram (-)cocci in pairsgram staining

Staphylococcus aureusgram (+)cocci in clusters gram staining

Salmonella typhosagram (-)short bacilli arrange singlygram staining

Diplococcus pneumoniaegram (+)cocci in pairsgram staining

Vibrio choleraComma bacillusgram (-)curved bacilli, comma shapedgram staining

Spirillum volutansgram (-)spiral shapedgram staining

Escherichia coliColon bacillusgram (-)short bacilli arranged singly gram staining

Bacillus subtilisgram (+)bacilli in chains gram staining

Escherichia coligram (-)bacilli in singles

Sarcina luteagram (+)cocci in groups of 8gram staining

Mycobacterium tubercolosisTubercle bacilli / Koch’s bacillusAcid fastSlender bacilli in serpentetive cord patternZiehl Neelsen acid fast stain

non- acid fastcocci in tetrads, chains

EXERCISE 6 MOTILITY OF BACTERIA

Hanging Drop Preparation- Examine microscopically- Concavity slide/ depression slide- Materials: Vaseline or white petroleum jelly,

applicator slideBrownian movement: bombardment of molecules of water

Motile: Bacillus subtilisNon motile: Staphylococcus aureus

EXERCISE 7: ANTIBIOTIC SUSCEPTIBILITY TESTING

ANTIBIOTIC SUSCEPTIBILITY TESTINGPlated medium (Erlenmeyer flask 200 mL)

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

S. aureus specimen AE.coli specimen B

- Important in management of infectious diseases particularly if susceptibility pattern of microorganisms cannot be predicted

Manner of ReportingSusceptible/ Sensitive – growth is inhibited in vitro , effective against bacteriaResistant- 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 macroscopically1000 - turbid, not effective

Agar Dilution- Uses petri dish- Different concentration of chemotherapeutic

agents- Heated agar (not solidify yet)

Consider:

Plating mediumdepth of medium

- 4mm high- Too thick – false resistant- Too thin – false susceptible

Size of inoculum- Compare sa 0.5 MacFarland

allow to solidify then streak, incubate 18

-24 hrs, observe colonies

pH of medium- 7.2 – 7.4

Disk Diffusion- Kirby Baurer- MHA 1. Batch to batch uniformity 2. Low in

sulfonamide and tetracycline inhibitors - Streaking : overlapping using sterile cotton

swabs - Don’t place 24 mm near center- Don’t 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 sa reference - 6mm measurement of antibiotic disk

EXERCISE 8 BACTERIA OF THE RESPIRATORY TRACT, THROAT CULTURE

Blood agar plate – 28 g / LMannitol salt agar plate – 111 g / LChocolate agar plate – 28 g/LPhenylethyl alcohol agar – 35.5 g/LEosin methylene blue – 36 g / L

BAP

Pinhead, creamy white to yellow, convex, smooth glistening colonies

β hemolytic: complete with clear zone ( pathogenic Staphyloccoci)

δ hemolytic – no zone of hemolysis ( non- pathogenic Staphyloccoci)

Pinpoint, flat gray translucent colonies

β hemolytic: complete with clear zone ( pathogenic Staphyloccoci)

α hemolytic – incomplete greenish zone of hemolysis

δ hemolytic – no zone of hemolysis ( non- pathogenic Staphyloccoci)

Large, gray mucoid colonies

With or without hemolysis ( gram – enteric bacilli)

Large swarming, spreading colonies with mousy or burnt chocolate odor

Proteus species (urine culture only)

CAP

Pinhead, creamy white to yellow colonies

With greenish discoloration ( pathogenic Staphyloccoci)

w/out greenish zone ( non- pathogenic Staphyloccoci)

Pinpoint, flat, gray colonies

With greenish discoloration (β hemolytic Streptoccoci)

w/out greenish discoloration ( α, δ hemolytic Streptococci)

Large mucoid with or without greenish discoloration

Gram (-) enteric bacilli

PEApinhead pinpoint

- Catalase test- Mannitol

fermentation test- Coagulase slide

method- If (-), do coagulase

tube method- Make a smear

- Catalase- Make a smear

EMB

Pink violet coloniesLactose fermenting gram (-) bacilli

Colorless coloniesNon lactose fermenting gram (-) bacilli

Catalase test - Colonies on slide- 2 drops of 3% hydrogen peroxide- (+) bubbles

PATHOGENECITY TEST FOR STAPHYLOCOCCI

A. Coagulase test1. 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

MSAYellow colonies Mannitol fermenting

staphylococci speciesPink colonies Non- mannitol fermenting

staphylococci species

EXERCISE 9: BACTERIA OF UROGENITAL TRACT (URINE CULTURE)

MAC

Pink violet coloniesLactose fermenting gram (-) bacilli

Colorless coloniesNon lactose fermenting gram (-) bacilli

- Multiply 1000 to get CFUs/ml- Gram (-) bacilli, singles