Test: Gram StainPurpose: determine gram reaction of bacteria. Will either be gram negative or gram positive. Serves a dual function: as a protein dehydrating agent (decreases pore size) and as a lipid solvent. It’s action is determined by the thickness of the peptidoglycan layer and the concentration of lipids on the surrounding the cell wallMethod: 1. Heat fix- add drop of water to slide and add bacteria, allow to air dry, then heat fix.3. Add crystal violet stain for 1 minute, wash w/water, all cells appear purple4. Add Gram’s Iodine for 1 minute, wash w/water, 5. decolorize with ethanol/acetone. Let it trickle down the slide until run off is clear- No more than 10 seconds. Rinse with water, but do it FAST after ethanol6. counter stain with safranin for 1 minute, rinse with water Notable bacterial results: G- cells will be pink, G+ purple

G- G+Gram negative family includes Shigella, Salmonella, Proteus, Klebsiella,Escherichia,Enterobacter etc. Usually four tests are used for differentiation of the various members of Enterobactericeae. They are Indole test,Methyl red test, Voges proskauer test and Citrate test; collectively known as IMViC series of reactions.

strep

Staph

Bacillus/Rod

Vibrio

Spirila (rigid)

Spirochete (flexible)

Test: Phenylethyl Alcohol AgarPurpose: Isolation of G+ cocci- streptococci, enterococci, or staphylococci.Method:

1. Divide bottom of plate into 3 sections, label with organism, name and date.2. Mix cultures well, and spot inoculate 3. Invert and incubate 24-48 hrs.

Notable bacterial results: poor growth or no growth- probably gram negative. Good growth- probable staphylococcus, streptococcus, enterococcus.

Test: Manitol Salt AgarPurpose: isolation and differentiation of staphylococcus aureus. Manitol provides the substrate for fermentation and makes the medium differential. Sodium chloride makes medium selective because it concentration is high enough to dehydrate and kill most bacteria. Phenol red indicates whether fermentation has taken place by changing color as pH changes.Method:

1. divide plate into 3 and label with organism, name, date.2. Mix culture well and spot-inoculate3. Invert and incubate the plate for 24-48 hrs.

Notable bacterial results: poor growth or no growth- not staphylococcus good growth- staphylococcus yellow growth or halo-possible pathogenic staphylococcus aureus red growth (no halo)- stephlyococcus other than S. aureus

Test: MacConkey AgarPurpose: selective and differential medium containing lactose, bile salts, natural red, and crystal violet. Used to isolate and differentiate members of the Enterobacteriaceae based on the ability to ferment lactose. Gram negative selection.Method:

1. divide bottom of plate into three sections and label.2. Mix each culture well and spot-inoculate the sectors with unknowns.3. Invert and incubate for 24-48 hours.

Notable bacterial results: Poorgrowth or no growth- G+ Good growth- G- Pink to red growth with or without bile precipitate- coliform probably E. coli or

Enterobacter Growth is not red or pink- noncoliform Coliform bacilli :

lactose fermenters (produce acid), exhibit a red color on their surface

Test: Eosin Methylene Blue AgarPurpose: selective and differential medium that contains peptone(carbon, nitrogen, and other nutritional components), lactose (supports coliforms such as E. coli), sucrose(supports Proteus or Salmonella) and the dyes eosin Y and methylene blue (inhibit G+ growth, and react with lactose fermenters turn the growth dark purple or black)Method:

1. Divide plate into sections and label.2. Mix each culture well3. Spot inoculate4. Invert and incubate the plate for 24-48 hours.

Notable bacterial results: Poor growth or no growh- inhibited by methylene blue-G+ Good growth- not inhibited by methylene blue-G- Growth is pink and mucoid- ferments lactose with little acid production- possible

coliform Growth is dark (purple to black with or without green metallic sheen)-ferments

lactose and/or sucrose with acid production-probably coliform Growth is colorless (no pink, purple, or metallic sheen)-does not ferment lactose or

sucrose-no reaction-noncoliform

Test:Phenol Red BrothPurpose: Differential test medium used to tests an organism's ability to ferment the sugar glucose as well as its ability to convert the end product of glycolysis, pyruvic acid into gaseous byproducts. PR broth is used to differentiate members of Enterobacteriaceae and to distinguish them from other G- rods.Method:

1. Obtain four PR glucose broths. Label each with name, your name , medium name, and date

2. Inoculate each brother with a test organism3. Incubate tubes for 48 hours.

Notable bacterial results: Yellow broth, bubble in tube- +fermentation with acid and gas end products Yellow broth, no bubble in tube- fermentation with acid end products; no gas

produced Red broth, no bubble in tube- No fermentation Pink broth, no bubble in tube- degradation of peptone; alkaline end products

Test: TSI- Triple sugar iron agarPurpose: Differentiates bacteria on basis of glucose, lactose, and sucrose fermentation and sulfure reduction.Method:

1. Obtain slants and label with name, date, and unknown.2. Stab the butt, and streak the slant.3. Incubate aerobically for 24 hours.

Notable bacterial results: Yellow slant/yellow butt-glucose and lactose and/or sucrose fermentation with acid

accumulation in slant and butt Red slant/yellow butt- glucose fermentation with acid production. Proteins

catabolized aerobically in the slant with alkaline products (reversion) Red slant/red butt- no fermentation. Proteins catabolized aerobically and

anaerobically with alkaline products. Not from Enterobacteriaceae. Red slant/ no change in the butt- no fermentation proteins catabolized aerobically

with alkaline products. Not from Enterobacteriaceae. No change in slant/ no change in butt- organism is growing slowly or not at all. Not

from Enterobacteriaceae. Black precipitate in the agar-sulfur reduction. (an acid condition, from fermentation

of glucose or lactose, exists in the butt even If the yellow color is obscured by the black precipitate)

Result (slant/butt) Interpretation

1 Red/Yellow Glucose fermentation only, peptone catabolized.2 Yellow/Yellow Glucose and lactose and/or sucrose fermentation.3 Red/Red No fermentation, Peptone catabolized.4 Yellow/Yellow with bubbles Glucose and lactose and/or sucrose fermentation, Gas produced.5 Red/Yellow with bubbles Glucose fermentation only, Gas produced.6 Red/Yellow with bubbles and black precipitate Glucose fermentation only, Gas produced, H2S produced.

7 Yellow/Yellow with bubbles and black precipitate Glucose and lactose and/or sucrose fermentation, Gas produced,H2S produced.

8 Red/Yellow with black precipitate Glucose fermentation only, H2S produced.9 Yellow/Yellow with black precipitate Glucose and lactose and/or sucrose fermentation, H2S produced.

Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae are lactose fermenters.

Salmonella typhimurium, Shigella dysenteriae, Proteus vulgaris, Pseudomonas aeruginosa, Alcaligenes faecalis etc are non lactose fermenters

Test: Methyl Red and Voges-Proskauer Tests (MRVP)Purpose: Combination medium used for MR and VP tests. Contains peptone, glucose, and a phosphate buffer. The peptone and glucose provide protein and fermentable carbohydrate, and the potassium phosphate resists pH changes in the medium. Method:

1. Obtain MR-VP broths inoculate with test cultures. 2. Incubate 48 hours. 3. Lab two- add 15 drops of VP reagent A. mix well. 4. Add 5 drops reagent B. mix well.5. Place on rack and observe for red color after 10 mins. (+) are pink and (-) are

copper.Notable bacterial results:

To differentiate: E. coli, E. aerogenes, K. pnemoniae Ferment glucose but quickly convert acid end products to intermediate (acetonin)

and 2,3-butanediol (end product) Produces red color when reacts with guanidine (peptone) in medium Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae are lactose

fermenters. Salmonella typhimurium, Shigella dysenteriae, Proteus vulgaris, Pseudomonas

aeruginosa, Alcaligenes faecalis etc are non lactose fermenters

When methyl red is added to MR-VP broth that has been inoculated with Escherichia coli , it stays red. This is a positive result for the MR test.

When methyl red is added to MR-VP broth that has been inoculated with Enterobacter cloacae , it turns yellow.  This is a negative MR result.

When the VP reagents are added to MR-VP broth that has been inoculated with Escherichia coli , the media turns a copper color. This is a negative result for the VP test.

When the VP reagents are added to MR-VP broth that has been inoculated with Enterobacter cloacae , the media turns red. This is a positive VP result.

http://www.austincc.edu/microbugz/mrvp_test.php

Test: Citrate TestPurpose: Nutrient utilization test. Contains sodium citrate as the only carbon-containing compound and ammonium ion as the only nitrogen source. Only organisms able to produce the enzymes specific to these compounds will be able to survive and grow on the medium. Method: Obtain three simmons citrate tubes. With a needle, streak the slants with the organisms. Incubate 48 hours.Notable bacterial results:

Positive=blue medium with growth Negative=green medium with no growth Enterobacter aerogenes (+) growth and blue Escherichia coli (-) no growth or change

Test: SIM MediumPurpose: used to determine sulfur reduction, indole production from trypophan, and

motility. Two Iron containing compounds: Organic Iron Source- Cysteine that is broken down by cysteine desulfuraseInorganic Iron Source- Sodium Thiosulfate, final electron acceptor in anaerobic ETC. Hydrogen sulfide produced in both reactions.Method: use Simmon tubes. Label and stab-inoculate to within 1 cm of the bottom of the tube. Incubate for 24-48 hours.Notable bacterial results:

Black in medium=sulfur reduction Red in the alcohol layer= tryptophan broken down into indole and pyruvate (+) Growth radiating outward from the stab line=motility Salmonella is positive for both so black, and negative for indole production shigella negative for all Streptococcus pyogenes negative for both so no change E. coli is positive for indole production, so red on top. Has motility but no H2S. Proteus vulgaris is positive for indole production Almost all spiral bacteria and about half of the bacilli are motile, whereas essentially

none of the cocci are motile.

Test: Oxidase TestPurpose: Oxidase enzymes are important to the electron transport system during aerobic respiration. Cytochrome Oxidase oxidizes (adds oxygen to) reduced cytochomes enzymes which carry out electron transport The oxidase test aids in the differentiation in Neisseria and Pseudomonas species, which are oxidase-positive, and Enterobacteria, which are oxidase-negativeMethod: 1) add a few drops of oxidase reagent to a piece of filter paper. 2) using wood applicator, pick up pre-grown culture and rub into the filter paper. 3) watch for color change within 30 secondsNotable bacterial results:

If color change to violet or dark purple then (+) If colorless or light purple/pink then (-) E. coli (-) Pseudomonas aeruginosa (+) Staph aureus (-)

Test: Nitrate Reductase TestPurpose: The reduction of nitrates occurs in the absence of Oxygen. Reduction = transfer of electrons to another sourceOne-step reduction or Partial reduction- NO3- (Nitrate) + Nitrate Reductase = NO2 (Nitrite)Complete Reduction or Denitrification- NO3 = N2 (molecular nitrogen, a gas) Assimilatory Nitrate Reduction -NO2 (Nitrite) + Nitrite Reductase = NH3 (Ammonia)Method: Obtain nitrate broths. Inoculate. Incubate for 24-48 hours. Lab two: examine each tube for gas production. Add 8 drops each of reagent a and b, mix well and let stand for ten minutes. Check for positives. Add zinc to remaining tubes let stand for ten. Check for changes.Notable bacterial results:

Tube 1 = Red color, positive reactionTube 2 = control, no color changeTube 3 = inconclusiveTube 4 = inconclusiveTube 5 = gas produced, positive for denitrification

Tube 1= Red color, positive reactionTube 2 = control, red color because zinc reduced the nitrates, negative reactionTube 3 = red color, zinc reduced nitrate, negative Tube 4 = no color change, zinc did not reduce nitrates so we know nitrate was reduced previouslyTube 5 = already known to be positive

Test: Urea Hydrolysis TestPurpose: Product of decarboxylation of amino acids. Hydrolyzed to ammonia. Nutrients in urea broth:

Urea Minimal percentage of yeast extract Buffers to prevent alkalinazation Phenol red, pH indicator

Method: Obtain three urea broths. Label. Inoculate with heavy inocula from the test organisms. Incubate aerobically for 24 hours.Notable bacterial results:

Urease is produced by some microorganisms Especially useful in identifying Proteus vulgaris Other microorganisms may produce urease, the urease in Proteus species tends

to act faster Useful for differentiating Proteus species from other non-lactose-fermenting

enteric organisms Ammonia creates alkaline environment causing the media to turn pink (+) Negative test is orange Proteus vulgaris (+) e coli (-) Pink=positive

STAPH IDENTIFICATION

Test: Catalase Test (slide test)Purpose: used to identify organisms that produce the enzyme catalase.Method: Transfer a large amount of growth to a slide. Aseptically place one or two drops of hydrogen peroxide directly onto bacteria and immediately observe for bubble formation.Notable bacterial results:

Visible bubble production is a positive test Bacteria that produce catalase can be detected Staph aureus (+) Strep (-)

Test: DNA Hydrolysis Test (DNAse)Purpose: Used to distinguish serratia species (+) from enterobacter species, moraxella catarrhalis (+) from neisseria species, and staphylococcus aureus (+) from other staph speciesMethod: Using a pen, divide the DNase test agar plate into equal sectors. Label. Spot-inoculate three sectors with the test organisms and leave a control space. Invert and incubate 24-48.

Notable bacterial results: (+) test indicated by growth and a clearing around the organisms Staph aureus (+) Serratia marcescens (+) Enterobacter aerogenes -? Top and bottom left on picture are +

Test: Novobiocin Susceptibility TestPurpose: Used to differentiate coagulase-negative staphMethod: Obtain blood agar plate. Cotton applicator to inoculate half of the plate for each. Allow it to absorb for 5 minutes. Sterilize forceps and put disc on plate. Invert and incubate 24-48.Notable bacterial results:

Staph epidermis- sensitive - (large zone) Staph saprophyticus- resistant (zone is under 16 mm)

resistant (saprophyticus) sensitive (epi)

Test: Coagulase TestPurpose: Used to differentiate staph aureus from other G+ cocci. Converts fibrinogen into fibrinMethod: Obtain slides and divide them in half. Add a drop of coagulase plasma and inoculate. Observe for agglutination.Notable bacterial results:

Thickness and clumping show a positive test Staph aureus (+) Staph epidermis (-)

STREP IDENTIFICATION

Test: Bile Esculin TestPurpose: Used for presumptive identification of enterococci and members of the strep bovis group, all of which are (+)Method: label & Inoculate two Bile Esculin slants. Incubated for 48 hours, any blackening before then should be considered a positive resultNotable bacterial results:

Bile + will darken media Many G- tolerate bile

Test: Bacitracin Susceptibility TestPurpose: Tests for sensitivity to the antibiotic bacitracin.Method: Blood agar plate. Cotton applicator to inoculate half of the plate for each. Allow to absorb for 5 mins. Sterilize forceps and put disc on plate. Invert and incubate 24-48 hours.Notable bacterial results:

Group A Strep will display a zone of inhibition around the disc Staph aureus resistant (top on picture)

Test: Blood AgarPurpose: Media contains 5% Sheep Blood & Tryptic Soy AgarAllow differentiation of organisms based on ability to hemolyze red blood cells (RBCs)Method:

1. Inoculate one Blood Agar Plate2. Divide one plate into four quadrants3. Streak a single line for each organism.4. Place in incubator

Notable bacterial results: Alpha, beta and gamma hemolysis Strep pyogenes beta hemolysis (complete lysis)

Strep pnemoniae- alpha hemolysis (incomplete lysis)

Strep epidermis- no hemolysis- gamma

Test: CAMP TestPurpose: Group B strep produce a peptide known as CAMPCAMP acts in concert with hemolysis produced by Staphylococcus aureus, increasing the hemolytic effect appearing as an arrow shaped zoneMethod: Obtain blood agar plate. Label. Single streak of staph a along one edge. Inoculate each with a dense smear of other orgs across from the s. aureus streak. Finish with a single streak from strep. Label. Invert. Incubate for 24.Notable bacterial results:

Streaked on blood agar, an arrowhead-shaped zone of hemolysis forms and is a positive result

Strep agalactiae (+)

Staph aureus (-)

Organisms List

1. Escherichia coli- gram neg under microscope- gram neg rod, pink on blood agar-small flat grey- smell like moth balls indole will be indole positive, catalase

2. Klebsiella pneumonia-blood agar similar to ecoli, more round looks sticky & wet, white, gram neg-pink rod shape, indole negative-will be pink with spot test

3. Bacillus cereus-gram negative, look like big fuzzy mold4. Salmonella typhimurium – gram neg, close to e-coli5. Shigella sonnei-salmonella looking, e-coli looking not likely6. Alcaligenes faecalis-gram neg rod-pink rod, hemolysis-zoning white almost clear,

oxidase positive 7. Citrobacter freundii- poop smell, gut intestines, gram neg, flat gray colonies, indole

negative, oxidase negative8. Enterobacter aerogenes- gram-neg, white color, wet looking, oxidase negative, indole

negative9. Pseudomonas aeruginosa- clear zoning underneath, green*, produce shiny metallic,

smell like grapes10. Proteus vulgaris- gram neg, lawn of bac, spread, gray film over plate, indole positive,

oxidase negative, will look very different spread colony**11. Staphylococcus epidermidis- gram positive, white, flat, dry,12. Enterococcus faecalis- neg blue small colonies gram positive, don’t have

hemolysis,blueish gray13. Staphylococcus aureus-grapes sometimes pairs14. Staphylococcus saprophyticus15. Streptococcus mitis-a lot of hemolysis16. Streptococcus pyogenes-whole plate clear17. Streptococcus agalactiae-green dark gray, small flat colonies, concave, fastidious

gram positive

Pink-negIndole-pink

Coagulase-staphEcoliProtues PseudoStaph strep

1-10 G- tests for them: catalase, MSA, BAP, bacitracin, camp, bile esculin, nitrate, coagulase11-17 G+ tests for them: oxidase, TSI, MVP, nitrate, macconkey, citrate, urease, SIM

FLOWCHART UNKNOWN G

Gram stain

Gram negative Rod

Oxidase test (positive)

Positive

Citrobacter freundii Enterobacter aerogenes Escherichia coliKlebsiella oxytoca Klebsiella pneumoniae Pseudomonas aeruginosa Pseudomonas aureofaciens

Indole test ( Positive)

Positive

Escherichia coli Klebsiella oxytoca

Negative

Proteus vulgaris Proteus mirabilis Serratia marcescens Morganella morganii

Negative

Citrobacter freundii Enterobacter aerogenes Klebsiella pneumoniae

Citrate Test (negative)

Positive Negative

Klebsiella oxytoca Escherichia coli

Motility Test (positive)