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 Coagulation of protein Culture media Apparatus Standard Condition

1. Autoclaving / steam
under pressure

Bacillus stearothermophilus - detect effectivitiy of autoclave - strip turns black Coagulation of protein Kills only vegetative not spores Dental instruments. Feeding bottles Coagulation of protein Culture media that can’t withstand autoclave

Autoclave

121°C 15- 20 mins 15 psi

100°C 10-20 mins boiler *timed when it starts to boil

2. Boiling 3. Fractional/ tyndalization/ interminent

100°C for 30 mins for 3 consecutive days with incubation Arnold’s sterilizer *to allow bacteria to germinate and to be killed the following day

4. Inspissation

Germination – spores transformed into vegetative cell then destroyed - Coagulation of protein - Used with high protein media (which can’t stand autoclaving) - Dorset egg medium, Loffler serum, Lowenstein jensen HTST – high temp short time LTH- low tem holding

Inspissator

75°C - 80°C 2 hrs for 3 consecutive days

72°C 15 s 5. Pasteurization UHT- ultra high temp 72-140-72°C 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 Apparatus Oven Standard Condition 160-180°C for 1.5 – 2 hours pasteurizer 60-63°C 30 mins

2. Open flame 3. Incenaration 4. Cremation -

Bacillus subtilis var. Niger 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

B. CHEMICAL METHODS OF CONTROL : DISINFECTANT AND ANTISEPTICS Sterilization – process of killing or destroying microorganisms and microbial spores Fungi- fungicidal Spores- sporicidal Virus- 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 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

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 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

BAP- Blood Agar Plate CAP- Chocolate Agar Plate Slant 6 screwcap/ group 5ml/ tube

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

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

4. Selective- incorporated, not alter growth of
undesired microorganisms, with inhibiting salts CLASSIFICATION OF CULTURE MEDIA A. According to physical state / consistency 1. Liquid- no solidifying agent (ex. visible growth of bacteria; nutrient broth becomes turbid) 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

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- 38°C solidifies, 100°C 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

TYPES OF CULTURE

1. Pure culture- contains one species of
microorganism

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

- 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 simple Tubed- 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°C 3. Get from 3rd quadrant 4. Plated- radial simple Tubed- butt, butt slant, slant, broth EXERCISE 4: CULTIVATION OF BACTERIA, MICROBES IN THE ENVIRONMENT Whole colony: punctiform circular rhizoid irregular Surface: smooth, glistening rough wrinkled dry, powdery Edge: entire undulate lobate curled Elevation: flat raised convex pulvinate umbonate size: small pinpoint pinhead large EXERCISE 5: STAINING METHODS: A. SIMPLE STAIN

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)

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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

EXERCISE 5: COMPOUND MICROSCOPE: FOCUSING Pseudomonas aeruginosa gram (-) short bacilli arranged singly gram staining

B. DIFFERENTIAL STAIN: GRAM STAIN
1. 2. 3. 4. Smear Crystal violet (1 min), wash water Gram’s 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 carbolfuchsin 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 Schaeffer’s 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

Diplococcus pneumoniae gram (+) cocci in pairs gram staining

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

Motile: Bacillus subtilis Non motile: Staphylococcus aureus Spirillum volutans gram (-) spiral shaped gram staining EXERCISE 7: ANTIBIOTIC SUSCEPTIBILITY TESTING ANTIBIOTIC SUSCEPTIBILITY TESTING Plated medium (Erlenmeyer flask 200 mL) - Sensitivity testing - Mueller Hinton Agar - Conc – 38 g / L x 200 mL Escherichia coli Colon bacillus gram (-) short bacilli arranged singly gram staining S. aureus specimen A E.coli specimen B Important in management of infectious diseases particularly if susceptibility pattern of microorganisms cannot be predicted

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

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 Agar Dilution - Uses petri dish - Different concentration of chemotherapeutic agents - Heated agar (not solidify yet) allow to solidify then streak,  incubate 18 -24 hrs, observe colonies

Mycobacterium tubercolosis Tubercle bacilli / Koch’s bacillus Acid fast Slender bacilli in serpentetive cord pattern Ziehl Neelsen acid fast stain non- acid fast cocci in tetrads, chains EXERCISE 6 MOTILITY OF BACTERIA Hanging Drop Preparation - Examine microscopically - Concavity slide/ depression slide - Materials: Vaseline or white petroleum jelly, applicator slide Brownian movement: bombardment of molecules of water

Consider: Plating medium depth of medium - 4mm high - Too thick – false resistant - Too thin – false susceptible Size of inoculum - Compare sa 0.5 MacFarland

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 / 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 β hemolytic: complete with clear zone ( pathogenic Pinhead, creamy white to Staphyloccoci) yellow, convex, smooth glistening colonies δ hemolytic – no zone of hemolysis ( nonpathogenic Staphyloccoci) β hemolytic: complete with clear zone ( pathogenic Staphyloccoci) Pinpoint, flat gray translucent colonies α hemolytic – incomplete greenish zone of hemolysis δ hemolytic – no zone of hemolysis ( nonpathogenic Staphyloccoci) With or without hemolysis ( gram – enteric bacilli) Proteus species (urine culture only) Pinpoint, flat, gray colonies

With greenish discoloration (β hemolytic Streptoccoci) w/out greenish discoloration ( α, δ hemolytic Streptococci) Gram (-) enteric bacilli

Large mucoid with or without greenish discoloration

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

pinpoint

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Catalase Make a smear

Pink violet colonies Colorless colonies

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

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

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

EXERCISE 9: BACTERIA OF UROGENITAL TRACT (URINE CULTURE) MAC Lactose fermenting gram (-) bacilli Non lactose fermenting gram (-) bacilli

Pink violet colonies Colorless colonies

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