Bambam2017 Made by AMFValle UST-FMS SecD Batch 2017

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Edward Lester
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1 Made by AMFValle UST-FMS SecD Batch 2017

2 Procedure (from Broth Cultures): Sterilize Inoculating Loop. Hold it over the bunsen burner flame in a vertical position until it is red hot. Then, hold it away from flame and let it cool. With sterilized inoculating loop, fish a loopful of bacterial suspension and spread evenly on marked surface on slide. Spread smear gently to make a thin film. Let smear dry in air. Sterilize loop after use. Fix the smear briefly by passing slides through the flame, film side up, 3x.

Utilizes only ONE dye (+) charged stains so that it will bind to the (-) bact. cell wall Identify bacteria based on morphology Size Shape Arrangement Procedure: 1.

3 Utilizes only ONE dye (+) charged stains so that it will bind to the (-) bact. cell wall Identify bacteria based on morphology Size Shape Arrangement Procedure: 1. Flood smeared area with METHYLENE BLUE stain 2. Allow stain to stay for 1-2 minutes 3. Wash stain with gentle stream of tap water 4. Blot dry using sheets of blotting paper Credits to L. Viray for the pictures

4 Procedure: 1. Flood smeared area with CRYSTAL VIOLET stain (initial stain) for 1 minute 2. Pour off stain, wash with gentle flow of water 3. Apply GRAM s IODINE (mordant) for 1 minute, then rinse off with water 4. Decolorize using ACETONE-ALCOHOL solution or 95% ETHYL ALCOHOL for 20 seconds 5. Apply SAFRANIN (counter stain) for 30 seconds, then wash off with water Differentiates Gram(+) and Gram (-)

5 Gram (+) Gram (-) Credits to L.Viray for the pictures

6 For Acid Fast bacteria (Mycobacterium tb) Procedure (KINYOUN s METHOD): 1. Cover smear with Kinyoun s CARBOL FUCHSIN stain (initial stain) for 2-3 minutes, then rinse with tap water 2. Flood smear with 3% ACID ALCOHOL (decolorizer) for seconds, then rinse with tap water 3. Cover smear with METHYLENE BLUE (counter stain) for seconds, then rinse with tap water and blot dry Bacterium Background Pink Blue Credits to K. Erguiza SecB 2014 for the picture

7 Report as follows: Reporting Acid Fast bacilli in Sputum specimen No AFB in at least 100 fields 0 or (-) 1-9 AFB in 100 fields +n (report actual AFB count), Suggest repeat collection AFB in 100 fields AFB in at least 50 fields >10 AFB/field in at least 20 fields Bacterium Pink Background Blue Credits to K. Erguiza SecB 2014 for the picture

For Bacillus subtilis and other sporeformers Procedure: 1. Flood slide with 5% Aqueous solution of MALACHITE GREEN and heat to gentle steaming for 2-3 minutes. Avoid overheating and drying. 2. Pour off excess stain and rinse with water 3.

8 For Bacillus subtilis and other sporeformers Procedure: 1. Flood slide with 5% Aqueous solution of MALACHITE GREEN and heat to gentle steaming for 2-3 minutes. Avoid overheating and drying. 2. Pour off excess stain and rinse with water 3. Flood smear with SAFRANIN for 30 seconds 4. Rinse again with tap water and blot dry Spore Green Vegetative cell Pink Credits to L. Viray for the picture

For Klebsiella pneumoniae, etc. INDIRECT METHOD (Congo Red): Also called negative or relief stain 1. Using a sterile inoculating loop, fish out a small colony of K.

9 For Klebsiella pneumoniae, etc. INDIRECT METHOD (Congo Red): Also called negative or relief stain 1. Using a sterile inoculating loop, fish out a small colony of K. pneumoniae and mix it with a drop of CONGO RED STAIN and spread 2 cm diameter. 2. DO NOT HEAT 3. Cover the smear with 1 % ACID ALCOHOL until smear turns blue 4. Air dry Capsule colorless Background Blue Credits to K. Erguiza SecB 2014 for the picture

10 For Klebsiella pneumoniae, etc. INDIA INK Method 1. Place a loopful of bacteria on a slide 2. Add a small amount of India ink and immediately cover with coverslip to allow fluid to spread as a thin film Capsule colorless Background India Ink Credits to K. Erguiza SecB 2014 for the picture

11 Physical and Chemical Disinfectant

12 Different Methods of Streaking 1. Simple streaking 2. 4 quadrant streaking isolation of colony 3. Overlapping streaking sensitivity testing

13 KIRBY-BAUER method Disk Diffusion Method Introduced by William Kirby and Alfred Bauer in 1966 Consists of newly seeded lawn of bacterium in a nutrient medium (MUELLER-HINTON AGAR) and filter paper disks impregnated with antibiotics Culture is incubated for 16 to 18 hours However, in the procedure given in the manual, we incubate for hours at 35 degrees Centigrade Measure ZONE OF INHIBITION (diameter) Diameter of zone Rate of DIFFUSION

14 Can be reported as follows 1. Resistant no zone of inhibition 2. Intermediate 3. Susceptible large zone of inhibition Credits to K. Erguiza SecB 2014 for the picture

15 Credits to K. Erguiza SecB 2014 for the picture

16 Differentiated by hemolysis, mannitol salt agar growth, and coagulase test S. aureus Deep gold to pale cream or white colonies Clear zone of hemolysis S.epidermidis Porcelain white colonies Non-hemolytic Credits to K. Erguiza SecB 2014 for the picture

17 CATALASE TEST (slide method) 1. Place a drop of 3% H2O2 (hydrogen peroxide) on slide 2. Get a loopful of colony, mix with the drop of 3% H2O2 on the slide 3. Observe for bubble formation (gas liberation) (+) Staphylococcus (-) Streptococcus Credits to K. Erguiza SecB 2014 for the picture

Wrap plate and incubate for 18-24 hours 7.

18 MANNITOL FERMENTATION Test 1. Streak on the Mannitol Salt Agar plate 2. Observe color of colonies Yellow colonies = fermentation of mannitol 3. Wrap plate and incubate for hours 7.5% NaCl (Salt) inhibits growth of other organisms S. aureus yellow colonies S. epidermidis pink colonies Credits to K. Erguiza SecB 2014 for the picture

19 COAGULASE TEST I. Slide Method 1. Place 2 drops of sterile saline on slide 2. Pick a colony using loop, mix with the saline 3. Add 1 drop citrated fresh normal plasma 4. CLUMPING = positive! (+) S. aureus (-) S. epidermidis Credits to K. Erguiza SecB 2014 for the picture

Incubate for 37 deg C in water bath and read results at 4 hours and 24 hours if still negative 4.

20 COAGULASE TEST II. Tube Method 1. Get tube containing 0.5 ml citrated fresh plasma 2. Suspend colonies on tube 3. Incubate for 37 deg C in water bath and read results at 4 hours and 24 hours if still negative 4. COAGULATION (by tilting tube) = positive! 5. Often, a clot will stick to the bottom of the tube even if it is inverted (+) S. aureus (-) S. epidermidis Credits to K. Erguiza SecB 2014 for the picture

21 Differentiated by type of HEMOLYSIS TYPES OF HEMOLYSIS 1. Beta complete hemolysis Completely decolorized Hgb More marked when the plate has been incubated in candle jar (5-10% CO2) Streptococcus pyogenes 2. Alpha partial or incomplete hemolysis Green-brown color (reduced Hgb) S. pneumoniae and Viridans 3. Gamma non-hemolytic, may have slight discoloration in the medium Enterococcus fecalis Credits to K. Erguiza SecB 2014 for the picture

Gram (+) cocci in chains Streptococcus pyogenes Group

Streptococcus pneumoniae Group B, Alpha hemolytic,

agar plate (CAP) POSITIVE QUELLUNG REACTION Viridans

22 Gram (+) cocci in chains Streptococcus pyogenes Group A, Beta hemolytic, susceptible to bacitracin Streptococcus pneumoniae Group B, Alpha hemolytic, lancet-shaped, susceptible to optochin in chocolate agar plate (CAP) POSITIVE QUELLUNG REACTION Viridans streptococci Alpha hemolytic Credits to K. Erguiza SecB 2014 for the picture

23 TOLERANCE to 6.5% NaCl 1. Inoculate to a tube of 6.5% NaCl broth (Brain Heart Infusion Broth supplemented with NaCl to 6.5% concentration) 2. Incubate at 35 deg C for hrs 3. TURBID = positive growth! (+) Group D Strep i.e. Enterococci

24 CATALASE TEST 1. Place a colony on a clean glass slide 2. Add a drop of 3% H2O2 3. Observe for bubble formation Credits to K. Erguiza SecB 2014 for the picture

25 Test for Susceptibility (Bacitracin & Optochin) 1. Place the disk on the blood agar culture 2. Incubate at 35 deg C for hours 3. Zone of Inhibition = Susceptible! BACITRACIN TEST (TAXO A) Susceptible: Group A Strep i.e. S. pyogenes OPTOCHIN TEST Susceptible: Group B Strep i.e. S. pneumoniae

Urethral smear: Gram negative diplococci with adjacent sides flattened (kidney-bean shaped) Usually found inside pus cells Intracellular in acute gonorrhea, in early infections

26 Urethral smear: Gram negative diplococci with adjacent sides flattened (kidney-bean shaped) Usually found inside pus cells Intracellular in acute gonorrhea, in early infections or in chronic gonorrhea But they may also be found extracellularly Modified THAYER MARTIN agar culture Sugar Fermentation test to differentiate: N. gonorrhea and N. meningitides

27 Sugar Fermentation Test Both are Glucose fermenters Only N. meningitides is maltose fermenter Yellow (+) Red (-) Sugar N. menigitides N. gonorrhea Glucose + + Lactose - - Maltose + -

30 I. NON-SPOREFORMER BACILLI Corynebacterium diphtheria II. ANAEROBIC SPOREFORMER BACILLI Clostridium III. AEROBIC SPOREFORMER Bacillus Credits to K. Erguiza SecB 2014 for the picture

31 Mott Mycobacterium other than Mtb NTM Nontuberculous mycobacterium LOWENSTEIN JENSEN (LJ) Medium Culture for M. tuberculosis

International Union Against Tb and Lung Disease Reporting Acid Fast bacilli in Sputum specimen No AFB in at least 100 fields 0 or (-) 1-9 AFB in 100 fields

33 International Union Against Tb and Lung Disease Reporting Acid Fast bacilli in Sputum specimen No AFB in at least 100 fields 0 or (-) 1-9 AFB in 100 fields +n (report actual AFB count), Suggest repeat collection AFB in 100 fields AFB in at least 50 fields >10 AFB/field in at least 20 fields 2+ 3+

34 EMBA (Eosin Methylene Blue Agar) Selective and Differential Lactose fermenter greenish metallic sheen in reflected light and blue black center on transmitted light; may be mucoid with grayish brown centers Nonlactose fermenter translucent, colorless to slightly amber colored colonies

35 EMBA (Eosin Methylene Blue Agar)

36 MacConkey Agar Lactose fermenter large, red colonies surrounded by turbid zones Nonlactose fermenter colorless and translucent colonies

37 MacConkey Agar

38 MacConkey Agar

39 Biochemical Tests Procedure

40 A. TRIPLE SUGAR IRON Tests the ability to utilize glucose anaerobically with formation of acid and products (fermentation) Indicator: Phenol Red Basic Acidic Sugars Glucose/Dextrose Lactose Sucrose Tests: Sugar fermentation, H2S production, Gas production

41 A. TRIPLE SUGAR IRON Acid slant yellow Acid butt yellow Alkaline slant red Acid butt yellow Alkaline slant red Alkaline butt red Blackening of butt Cracks, bubbles or displacement of medium All sugars fermented GLUCOSE fermented No sugar fermented Produce H2S Produce gas (aerogenic)

B. LYSINE IRON AGAR Decarboxylation and Deamination Decarboxylation of amino acids (lysine, ornithine, arginine) resulting in alkalination of

42 B. LYSINE IRON AGAR Decarboxylation and Deamination Decarboxylation of amino acids (lysine, ornithine, arginine) resulting in alkalination of the media Indicator: Bromcresol purple Basic Acidic Tests: Deamination, Lysine decarboxylation Manner of Inoculation Stab the butt Streak the slant

43 B. LYSINE IRON AGAR Lysine Deamination (SLANT) Deamination occurs in the presence of O2, kaya sa slant part (exposed to O2) Deamination produces acidic species Red slant (+), acidic Purple slant (-), basic Lysine Decarboxylation (BUTT) Decarboxylation produces basic species (you remove the carboxyl end, natira si amino which is basic) Purple butt (+), basic Yellow butt (-), acidic

44 C. MOTILITY INDOLE ORNITHINE Indicator: Bromocresol Purple Motility Indole Ornithine Medium (MIO) Tests: Motility, Indole production, Ornithine decarboxylation Inoculation: Stab the butt For Motility and Decarb testing; 1. The portal of entry should be the portal of exit to eliminate false (+) results 2. Stab the medium up to the bottom because decarboxylation occurs at the deep butt 3. Do not shake. Medium is semisolid in consistency. 4. Read the motility and decarboxylation result before adding Kovac s Reagent!

around stabbing line (+) Decarboxylation = purple butt For Indole Production; Add 2-3 drops Kovac s reagent Tryptophanase:

45 C. MOTILITY INDOLE ORNITHINE Indicator: Bromocresol Purple Motility Indole Ornithine Medium (MIO) Tests: Motility, Indole production, Ornithine decarboxylation Inoculation: Stab the butt For Motility and Decarb testing; (+) Motile = diffuse growth around stabbing line (+) Decarboxylation = purple butt For Indole Production; Add 2-3 drops Kovac s reagent Tryptophanase: Tryptophan Indole (+) Indole Production = red ring upon addition of Kovac s modification of Erhlick s reagent (paradimethylaminobenzaldehyde)

D. CITRATE UTILIZATION TEST Tests ability to synthesize complex carbon compounds from an inorganic carbon source like citrate (+) for Autotrophs Many soil bacteria

46 D. CITRATE UTILIZATION TEST Tests ability to synthesize complex carbon compounds from an inorganic carbon source like citrate (+) for Autotrophs Many soil bacteria DO NOT have this ability Indicator: Bromothymol Blue Simmon s Citrate Agar Test: Citrate Utilization Test Inoculation: Streak the slant (+) Blue Slant (-) Green Slant

47 E. UREASE TEST Detects ability to split urea to ammonia (basic!) and CO2 by the enzyme urease Indicator: Phenol red Medium: Urea Broth Test: Urease Test (+) Fuschia Pink (-) Salmon Pink/Yellow

48 E. UREASE TEST

57 Selective for S. typhi

62 A. Water Collection 1. Clean the mouth of faucet with 70% EtOH 2. Collect 100 ml of chlorinated (tap water) by allowing water to flow about 1-5 minutes 3. Alternative: unchlorinated (deep well) water 4. Refrigerate while waiting to transport 5. Transport in ice (4 degrees Celsius)

63 B. Methods of Water Analysis 1. Heterotrophic Plate count: Pour Plate Method 2. Multiple Tube Fermentation Technique (MTFT) for Coliform group of organisms a) Presumptive test b) Confirmatory test c) Completed test

64 1. Heterotrophic Plate Count: Pour Plate Method

65 MTFT: Presumptive test Medium: Triple Strength Lauryl Tryptose Broth Incubation: 24 hours 35 deg C (+) Coliform bacteria (+) Gas formation and turbidity

66 MTFT: Confirmatory Test Medium: Brilliant Green Lactose Broth (BGLB) Incubation: 48 hours 35 deg C (+) Total coliforms

67 MTFT: Confirmatory Test Medium: EC Broth Incubation: 24 hours 44.5 deg C deg C (+) Fecal coliforms

69 Computation of MPN (Most Probable Number)

70 Reviewer by Bambam2017

320 MBIO Microbial Diagnosis. Aljawharah F. Alabbad Noorah A. Alkubaisi 2017

320 MBIO Microbial Diagnosis. Aljawharah F. Alabbad Noorah A. Alkubaisi 2017 320 MBIO Microbial Diagnosis Aljawharah F. Alabbad Noorah A. Alkubaisi 2017 Primary Media for Isolation of Microorganisms As we know, many clinical specimens contain a mixed flora of microorganisms. Thus