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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 cant withstand autoclave
Autoclave
100C for 30 mins for 3 consecutive days with incubation Arnolds 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 cant stand autoclaving) - Dorset egg medium, Loffler serum, Lowenstein jensen HTST high temp short time LTH- low tem holding
Inspissator
72C 15 s 5. Pasteurization 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 Apparatus Oven Standard Condition 160-180C for 1.5 2 hours pasteurizer 60-63C 30 mins
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
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
BAP- Blood Agar Plate CAP- Chocolate Agar Plate Slant 6 screwcap/ group 5ml/ tube
5ml x 6= 30 = 70 ml
Butt -
3ml x 7= 21 ml = 50 ml
TYPES OF CULTURE
- 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, 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 1. Plated medium (clock method) 2. Incubate for 18- 24 hrs at 37C 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
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
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
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
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
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 / Kochs 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 - 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 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 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 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) Pinpoint, flat, gray colonies
With greenish discoloration ( hemolytic Streptoccoci) w/out greenish discoloration ( , hemolytic Streptococci) Gram (-) enteric bacilli
PEA pinhead Catalase test Mannitol fermentation test Coagulase slide method If (-), do coagulase tube method Make a smear
pinpoint
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