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


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


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

Incenaration 4. Niger Burning to ashes Needles. Open flame 3. Cremation - Bacillus subtilis var.2. infected animals. wound dressings Cremate bodies with HIV Bunsen burner Incinerator . loops. inoculating needle (red hot) Burning to ashes Waste products Sputum cups.

2 plates/ student . wet hands with warm water 2.Disruption of cell membrane Sepsis. throroughly clean between fingers.chemical agent that kills bacteria : vegetative cells only Antimicrobial agent / drug – chemical agent that kills / inhibits without damaging the body tissue Sanitizer.virocidal Bacteriostatic. Metabolic elements Plated (500 mL Ernlenmeyer flask) 1.Material containing essential nutrients for growth of bacteria .Lipid dissolution . Dispense 4.Protecting self and microorganisms you are working on . Dissolving (hot plate) 3. rinse hands in downward position 6. Source of nitrogen 3. Formation (dispense in petri dish) Tubed (beaker) 1. Source of carbon 2.Adequate amount of water and salt .Protein denaturation Soap .Bacterial protein denaturation . dry with paper and hand towel 7.Contain essential nutrients in proper concentration .Protected by sterilization by UV light / passage of filters . turn off faucet with the unused paper towel to prevent contamination EXERCISE 2: PREPARATION OF CULTURE MEDIA CULTURE MEDIA . HAND SCRUBBING 1. Autoclave 6.Granular or powder form .Cell membrane destruction . CHEMICAL METHODS OF CONTROL : DISINFECTANT AND ANTISEPTICS Sterilization – process of killing or destroying microorganisms and microbial spores Fungi.20ml/ plate 20ml x 14= 280 = 300ml Butt slant .B.Cytoplasmic membrane destruction .10 ml/ tube 10ml x 6 = 60 = 100 ml .presence of bacteria in a system Asepsis. Source of minerals and vitamins 4.inhibits the growth of microorganisms Bactericidal – kills the growth of microorganisms Disinfection – inhibiting the growth of microorganisms Disinfectants.Protected from aerosols Filters: HEPA high efficiency particulate air filter ULPA ultra low particulate air filter C. apply antimicrobial soap 3.fungicidal Spores.6 loefflers/ group . Formation Plated .sporicidal Virus. dispensing 6.Proper pH .absence of bacteria in a system Biological safety cabinet . Dissolve 3. Autoclave 5. Weigh 2. create friction and loosen debris 4. under fingernails and rings and up to the wrist for atleast 15 seconds 5.agent that limits growth of bacteria to a safe level Antiseptic. Lysol Mechanism of action: Lysol .Free of inhibitory substance . Plugging (gauze) 4.Right moisture Nutrients required: 1. Weighing 2. rub to form lather.Sterile .Serves as food sand soil Criteria .Inactivation of enzymes Alcohol( 70% alcohol) .prevents growth of microorganisms by inhibiting their growth / activity ex. Plugging 5.

different culture media Culture.process of implanting/ transferring microbes/ infectious materials into culture media Materials: inoculating needle.incorporated. Synthetic/ chemically defined/ complex 2. Liquid.picking up of single colony for transferring into different culture media or smear 2.visible growth bacteria of deposited on the surface of solid media Fishing. Solid. 100°C liquefies solidifying agents. chloral hydrate.inhibits the growth of gram positive bacteria C.5-1% agar Gelatin.support growth of non-fastidious bacteria NB.albumin .prevents swarming of proteins K tellurite. cultivation and growth of bacteria and other species Inoculation.allows differentiation of 2 or more bacteria. According to function 1. NA.2ml/ tube 2ml x 3= 6 = 10 ml 4. beef extract. According to physical state / consistency 1. agar. pink.growth of microorganism on nutrient medium Colony.38°C solidifies.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 . incorporated is indicator Mannitol Salt Agar Phenol indicator Yellow halo colonies (Staphylococcus aureus) Red / pink colonies (Staphylococcus epidermis) Mac Conkey Agar Indicator. nutrient broth becomes turbid) Alcohol. Basal/simple/ordinary Basic.3 wasserman/ group . According to composition 1.no solidifying agent (ex. 0. Semi-solid.used for motility test.neutral red Lactose fermenters: red. According to form 1.blood.general culture media Composition of simple solid mediumpeptone. sodium desoxycholate. gelatin B. Non-synthetic/ chemically undefined D. tubed EXERCISE 3: TRANSFER OF BACTERIA: ASEPTIC TECHNIQUE Microbial techniques.5 – 3% solidifies by using red algae polysaccharide) 3. Enriched. with inhibiting salts CLASSIFICATION OF CULTURE MEDIA A. agar 2. serum. visible growth of bacteria.contains agar (1. not alter growth of undesired microorganisms. purple Non.Blood Agar Plate CAP.Chocolate Agar Plate Slant 6 screwcap/ group 5ml/ tube 3. plated 2. water.inhibits growth of gram negative bacteria Gentian violet.diagnostic test Agar. Na azide.used to support growth of fastidious bacteria ( difficult to grow) NA + enriching salts.BAP. Selective. Differential.methods employed for study. inoculation loop. bile salts. ascetic fluid .

TYPES OF CULTURE 1.Rotate.contains 2 or more species of 3.Roate 90°. streak Simple Clock method METHODS OF INOCULATING MICROORGANISMS IN TUBED 1.radial simple Tubed.by stabbing and by streaking ( zigzag motion) 3.Fish out colony from plate using inoculating needle .use of inoculating needle .butt. Butt . powdery Edge: entire undulate lobate curled Elevation: flat raised convex pulvinate umbonate size: small pinpoint pinhead large EXERCISE 5: STAINING METHODS: A. Mixed culture. microogranims Ex. broth 2. incubate for 24 hrs. MICROBES IN THE ENVIRONMENT Whole colony: punctiform circular rhizoid irregular Surface: smooth.by streaking 4.butt.24 hrs at 37°C Mixed culture.24 hrs at 37°C 3.use of inoculating needle and loop because there is no butt portion . Incubate for 18. glistening rough wrinkled dry. 18. slant. Broth . butt slant. Plated (clock method) . flame sterilize .Lines of streaking are parallel to each other but should not overlap with other lines.radial simple Tubed. Plated.flame sterilize.Rub side of the test tube until it becomes turbid 5. Get from 3rd quadrant 4. Plated medium (clock method) 2. flame sterilize . Pure culture. restreak.Stabbing the butt portion (1/4) 2. SIMPLE STAIN Radial. touch last two lines of the previous streak.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 . e. streak. butt slant. 37°C PRACTICAL: Pure culture 1. cover more than 1/3 of the surface.contains one species of microorganism 2. slant. Slant . 4. Butt slant .always start in plated medium 1. broth EXERCISE 4: CULTIVATION OF BACTERIA. Plated.

wash water Decolorize with acetone. safranin. Blot dry. methylene blue. Prepare and heat fix sputum 2. blot dry 5. malachite green 1. 2. Wash with dH2O. wash water Gram’s iodine (1 min). heat fix 3. Carbol fuchsin 3.club-shaped w/ barbed ends . 3. SELECTIVE STAINING: SPORE STAIN. Drop of water / NSS + small amount of growth using loop or needle 2. decolorize acid alcohol (15-20s). Counterstain methylene blue (1min) 6. Spread. OIO EXERCISE 5: COMPOUND MICROSCOPE: FOCUSING Pseudomonas aeruginosa gram (-) short bacilli arranged singly gram staining B. OIO Positive: Red Negative: Blue Corynebacterium diptheriae Kleb. OIO Positive: Purple Negative: Red C. Wash tap water . Counterstain with safranin (30 s) wash water 6. comma shaped gram staining . Wash with dH2O 5.- Crystal violet. DIFFERENTIAL STAIN: ACID FAST STAIN 1. CAPSULE STAIN Spore: Bacillus subtilis Fulton Schaeffer’s method dH2O – drop. 4. wash water 5. Wash with dH2O.Loefflers bacillus gram (+) irregular bacilli with Babes Ernst bodies . Stain for 1-2 minutes 4. X.V or chinese characters arrangement Gram staining Neisseria gonorrhoeae gram (-) cocci in pairs gram staining D. 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. Smear Crystal violet (1 min).blot dry. Water bath. adding more carbolfuchsin 4. DIFFERENTIAL STAIN: GRAM STAIN 1. Steam for 5 minutes.Y.alcohol/ 95% ethyl alcohol.

applicator slide Brownian movement: bombardment of molecules of water  Consider: Plating medium depth of medium .Heated agar (not solidify yet) allow to solidify then streak.5 MacFarland .Sensitivity testing . Disk Diffusion– Kirby Bauer Method Broth Dilution .1000 / 2  500 250 (conc. of antibiotic / chemotherapeutic agent) Observe macroscopically 1000 .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 . Dilutions – Broth /Agar dilution 2.turbid. aureus specimen A E.Different concentration of chemotherapeutic agents . effective against bacteria Resistant. not effective Agar Dilution .4mm high .acid fast cocci in tetrads. against growth of bacteria Intermediate Susceptibility test 1.Concavity slide/ depression slide . observe colonies Mycobacterium tubercolosis Tubercle bacilli / Koch’s bacillus Acid fast Slender bacilli in serpentetive cord pattern Ziehl Neelsen acid fast stain non. chains EXERCISE 6 MOTILITY OF BACTERIA Hanging Drop Preparation .Different concentration of chemotherapeutic agents by serial dilution.Uses petri dish .not effective.Conc – 38 g / L x 200 mL Escherichia coli Colon bacillus gram (-) short bacilli arranged singly gram staining S.Mueller Hinton Agar .Examine microscopically .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) . uses 2 fold dilutions .Too thin – false susceptible Size of inoculum .Compare sa 0.  incubate 18 -24 hrs.Materials: Vaseline or white petroleum jelly.Too thick – false resistant .

gray colonies With greenish discoloration (β hemolytic Streptoccoci) w/out greenish discoloration ( α.Human plasma + organism from BAP . Test tube – 0.6mm measurement of antibiotic disk EXERCISE 8 BACTERIA OF THE RESPIRATORY TRACT.Don’t place 24 mm near center .(+) bubbles PATHOGENECITY TEST FOR STAPHYLOCOCCI A.(+) clumping 2.Incubate.Composition 0. creamy white to Staphyloccoci) yellow.5 MacFarland ( 1. smooth glistening colonies δ hemolytic – no zone of hemolysis ( nonpathogenic Staphyloccoci) β hemolytic: complete with clear zone ( pathogenic Staphyloccoci) Pinpoint. 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.pH of medium .Kirby Baurer .Don’t place 10-15mm periphery .4 Disk Diffusion . Low in sulfonamide and tetracycline inhibitors .2 drops of 3% hydrogen peroxide . convex. Batch to batch uniformity 2. Slide method .Streaking : overlapping using sterile cotton swabs .mannitol fermenting staphylococci species Large. spreading colonies with mousy or burnt chocolate odor CAP With greenish discoloration ( pathogenic Pinhead. flat. compare sa reference .Zone of inhibition – measure diameter.7. Diluents – NSS/NB .1mL 75% BaCl2 99. Mannitol Fermentation Yellow colonies Pink colonies MSA Mannitol fermenting staphylococci species Non. more susceptible .5 g/L Eosin methylene blue – 36 g / L BAP β hemolytic: complete with clear zone ( 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 .5 ml human plasma + organism from BAP .MHA 1.5 mL 1% H2SO4) . clot 30 minutes B.If too thick. 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. δ hemolytic Streptococci) Gram (-) enteric bacilli Large mucoid with or without greenish discoloration - - PEA pinhead Catalase test Mannitol fermentation test Coagulase slide method If (-). do coagulase tube method Make a smear pinpoint - Catalase Make a smear Pink violet colonies Colorless colonies EMB Lactose fermenting gram (-) bacilli Non lactose fermenting gram (-) bacilli Catalase test . you need to dilute. gray mucoid colonies Large swarming. Coagulase test 1.Colonies on slide .2 – 7.

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

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