Microbiology 217

1. Clean slide-- Bon Ami
2. Target circle
3. Broth: 3 loops; Agar: 3 loops water + 1 needle cells
4. Air dry
5. Flame "fix" X3

*Basic dye = positive chromophore
*e.g.: Crystal violet, Me-blue, Saffranin.
*check for morphology, palisade srrangement, metachromatic granules (volutin) w/ Me-blue
1. Prepare smear w/ heat fix
2. Add stain for 40-75 sec.
3. Rinse w/ water 2-3 seconds or 'til clear.
4. View w/ oil

*Always done WITHOUT HEAT FIXING
*Acidic stain = negative chromophore
*e.g., India ink, nigrosin, Congo red.
1. Add 2-3 loops cells in broth to 1 small drop India ink
2. Spread cells with a slide
3. Air dry
*use to identify capsule, morphology, real size

*Basic stains = positive chromophore
1. Prepare smear
2. PRIMARY STAIN: Cover with piece of Kimwipe, flood with Malachite green. Steam for 5 min.
3. DECOLOURIZER: rinse w/ water 'til runs clear (30 sec)
4. COUNTER STAIN: Safranin 20 sec
5. RINSE: w/ water 'til clear (2-3 sec)
6. Blot & view
**Spores are light green, cells are pink/red
**Use for genera <i>Bacillus </i> and <i> Clostridia </i>

*Positive chromophores (Basic)
1. Prepare smear
2. PRIMARY STAIN: Flood w/ Carbol-fuchsin in phenol. Steam 5 min.
3. RINSE: w/ water 2 sec
4. DECOLOURIZER: ACIDIFIED alcohol for 40 sec or until runs clear.
4. COUNTER STAIN: Me-blue 30 sec
5. RINSE: w/ water 2 sec or 'til clear
6. Blot and view
**Mycobacteria & Nocardia (acid-fast) stain Red. All others (non-acid-fast) stain blue.

*Positive chromophores (Basic)
1. Prepare smear
2. PRIMARY STAIN: Crystal violet 20 sec
3. MORDANT: Grams iodine 1 min
4. DECOLOURIZER: alcohol 15 sec or 'til clear
5. RINSE: 2 sec
6. COUNTER STAIN: Safranin 1 min
7. RINSE: 2-3 sec or 'til clear
8. Blot & view

Fermentation of glucose
PREPARE DUPLICATE TUBES--one will test aerobic resp, the other will test anaerobic resp.
1. Transfer cells with needle
2. Aer: Leave tube as is. Anaer: Layer 1 ml mineral oil on top of tube
3. Incubate at 37 C for 24 hr, 48 hr.
4. Compare BOTH tubes:
BOTH Yellow:--> Aerobic & Fermentative resp occurs.
ANAER: Green, AER: Yellow--> Oxidative resp only
ANAER: Yellow, AER: Green --> Fermentative resp only
BOTH Green: ---> Sugar can't be metabolized.

1. Inoculate FTM medium with one loop of bacteria.
2. Mix cells w/ medium by rolling culture tube betwseen palms
3. Incubate 24 hr at optimum temp; or 48 hrs at optimum T-10C
**AEROBIC: growth @ top of tube
FACULTATIVE: growth throughout tube
ANAEROBIC: growth at bottom of tube
MICROAEROPHILIC: growth 1/3 down tube. These cells prefer low O2 (5-10%)

1. Inoculate SIM medium with a needle stab.
2. Incubate at 25C
**Turbid or diffuse medium indicates motility

Used to determine if cells can ferment a given sugar. A Durham tube collects respiratory gases, and a pH indicator (phenol red) shows acidic products.
1. Acquire an uniculatedf control tube.
2. Inoculate a second tube with a loop of cells.
3. iIcubate at 37C for 24-48 hrs.
**YELLOW: Acid products (fermentation)
GAS: (over 10% of Durham) CO2 or H2 by-products

Used to identify bacteria that produce a MIXTURE of acidic products, including lactic acid, formic acid and acetic acid. Ethanol, CO2 and H2 are also made.
**Used in identifying E coli-- which is positive for MR
**Medium contains Methyl Red indicator.
1. Inoculate a MR-VP tube with 1 loop of cells.
2. Incubate 3-5 days at 25C
3. Add 4 drops of methyl red indicator.
**RED colour indicates mixed acid fermentation.
NO CHANGE indicates a non-mixed-acid organism.
NB: Mixed-acid fermenters include Escherichia, Salmonella, Proteus & Aeromonas

Used to identify coliforms that produce 2,3-butanediol during fermentation (and are therefore less acidic than mixed acid fermenters)
**You must use 3-5 day old cultures to get good results!
1. Inoculate a VP tube with 1 loop of cells.
2. Incubate 3-5 days at 25C
3. Remove 1 mL of cells and place in an empty tube.
4. Add 0.5 mL Barritt's Reagent A (alpha naphthol) and mix.
5. Add 0.5 mL Barritt's Reagent B (KOH) and mix VIGOROUSLY.
6. Allow the tube to stand for 30 min.
**PINK/RED: Butanediol fermenter
NO CHANGE: nonbutanediol fermenter
NB: Butanediol fermenters include Klebsiella and Enterobacter

Identifies bacteria that produce AMYLASES and MALTASE, enzymes that digest starch.
1. Streak a single, wide swath of cells on a starch agar plate.
2. Incubate 24-48 hr at 37C.
3. Flood plate with Grams iodine for 30 sec, then pour off excess in biohazard bin.
** A CLEAR MARGIN around the streak indicates AMYLASE activity (positive). activity (positive). No clear margin is a negative test.

Identifies bacteria that produce LIPASE, an enzyme that digests starch.
1. Streak a single, wide swath of cells on a TRIBUTYRIN agar plate.
2. Incubate 24-48 hr at 37C.
** A CLEAR MARGIN around the streak indicates LIPASE activity (positive). No clear margin is a negative test.

Used to identify bacteria that use nitrogen compounds as electron acceptors.
**Some bacteria reduce nitrate to nitrite only. Others reduce nitrate to nitrite, and then reduce nitrite to N2 gas.
1. Inoculate a culture tube containing beef extract and potassium nitrate with a loop of bacteria.
2. Place an inverted Durham tube in the tube.
3. Incubate 2-4 days at 25C
4. Inspect the Durham tube for a gaseous fermentation product. Any gas produced must be from the reduction of nitrate, as no sugar is included in the medium.
5. Add 2-3 drops of Reagent A (sulfanilic acid)and 2-3 drops of Reagent B (dimethyl-alpha-naphthylamine) to any tube lacking a gasesous product.
**RED: Nitrate reduced to nitrite.
COLOURLESS:
EITHER No reduction, or the nitrite was further reduced to N2 gas. To determine which of these a colourless tube represents--
6. Add a pinch of zinc metal and shake vigorously.
RED: Negative test. (Zinc was reduced by the nitrate, yiuelding the red colour.)
COLOURLESS: Positive test. (Nitrate was reduced to nitrite, and then nitrite was converted to a non-gaseous product or N2 gas that escaped.)

Identifies bacteria that produce CASEASES, enzymeS that digests the milk protein, casein.
1. Streak a single, wide swath of cells on a SKIM MILK AGAR plate.
2. Incubate 24-48 hr at 37C.
** A CLEAR MARGIN around the streak indicates Casein hydrolysis (positive). No clear margin is a negative test.

The test identifies aerobic bacteria that use cytochrome c as their final electron acceptor.
*Used to distinguish (oxidase-positive) Pseudomonas from (oxidase-negative) Enterobacteriaceae.
1. Divide an agar plate in half.
2. Streak 1/2 of the plate with Pseudomonas aeruginosa, and the other half with an unknown bacteria.
3. Incubate at 37C for 48 hr.
4. Use a cotton swab to collect a sample of Pseudomonas cells.
5. Add a few drops of N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride to the swab.
6. Repeat, swabbing the unknown and adding a few drops of the reagent.
**YELLOW--> PURPLE within 30 sec: positive test.
YELLOW unchanged or after 30 sec: negative test.