Test: Gram Stain
Purpose: 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 wall
Method:
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 purple
4. 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 ethanol
6. counter stain with safranin for 1 minute, rinse with water
Notable bacterial results: G- cells will be pink, G+ purple
GG+
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 Agar
Purpose: 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 Agar
Purpose: 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-inoculate
3. 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 Agar
Purpose: 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 Agar
Purpose: 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 well
3. Spot inoculate
4. 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 Broth
Purpose: 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 organism
3. 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 agar
Purpose: 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)
1
2
3
4
5
6
Result (slant/butt)
Red/Yellow
Yellow/Yellow
Red/Red
Yellow/Yellow with bubbles
Red/Yellow with bubbles
Red/Yellow with bubbles and black precipitate
7
Yellow/Yellow with bubbles and black precipitate
8
9
Red/Yellow with black precipitate
Yellow/Yellow with black precipitate


Interpretation
Glucose fermentation only, peptone catabolized.
Glucose and lactose and/or sucrose fermentation.
No fermentation, Peptone catabolized.
Glucose and lactose and/or sucrose fermentation, Gas produced.
Glucose fermentation only, Gas produced.
Glucose fermentation only, Gas produced, H2S produced.
Glucose and lactose and/or sucrose fermentation, Gas produced,
H2S produced.
Glucose fermentation only, H2S produced.
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.
2.
3.
4.
5.
Obtain MR-VP broths inoculate with test cultures.
Incubate 48 hours.
Lab two- add 15 drops of VP reagent A. mix well.
Add 5 drops reagent B. mix well.
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 Test
Purpose: 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 Medium
Purpose: 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 Test
Purpose: 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-negative
Method: 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 seconds
Notable 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 Test
Purpose: The reduction of nitrates occurs in the absence of Oxygen. Reduction = transfer
of electrons to another source
One-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 reaction
Tube 2 = control, no color change
Tube 3 = inconclusive
Tube 4 = inconclusive
Tube 5 = gas produced, positive for denitrification
Tube 1= Red color, positive reaction
Tube 2 = control, red color because zinc reduced the nitrates, negative reaction
Tube 3 = red color, zinc reduced nitrate, negative
Tube 4 = no color change, zinc did not reduce nitrates so we know nitrate was reduced
previously
Tube 5 = already known to be positive
Test: Urea Hydrolysis Test
Purpose: 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
species
Method: Using a pen, divide the DNase test agar plate into equal sectors. Label. Spotinoculate 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 Test
Purpose: Used to differentiate coagulase-negative staph
Method: 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 Test
Purpose: Used to differentiate staph aureus from other G+ cocci. Converts fibrinogen into
fibrin
Method: 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 Test
Purpose: 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 result
Notable bacterial results:
 Bile + will darken media
 Many G- tolerate bile
Test: Bacitracin Susceptibility Test
Purpose: 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 Agar
Purpose: Media contains 5% Sheep Blood & Tryptic Soy Agar
Allow differentiation of organisms based on ability to hemolyze red blood cells (RBCs)
Method:
1. Inoculate one Blood Agar Plate
2. Divide one plate into four quadrants
3. 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 Test
Purpose: Group B strep produce a peptide known as CAMP
CAMP acts in concert with hemolysis produced by Staphylococcus aureus, increasing the
hemolytic effect appearing as an arrow shaped zone
Method: 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 agarsmall 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 mold
4. Salmonella typhimurium – gram neg, close to e-coli
5. Shigella sonnei-salmonella looking, e-coli looking not likely
6. 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 negative
8. Enterobacter aerogenes- gram-neg, white color, wet looking, oxidase negative, indole
negative
9. Pseudomonas aeruginosa- clear zoning underneath, green*, produce shiny metallic,
smell like grapes
10. 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 gray
13. Staphylococcus aureus-grapes sometimes pairs
14. Staphylococcus saprophyticus
15. Streptococcus mitis-a lot of hemolysis
16. Streptococcus pyogenes-whole plate clear
17. Streptococcus agalactiae-green dark gray, small flat colonies, concave, fastidious
gram positive
Pink-neg
Indole-pink
Coagulase-staph
Ecoli
Protues
Pseudo
Staph
strep
1-10 G- tests for them: catalase, MSA, BAP, bacitracin, camp, bile esculin, nitrate,
coagulase
11-17 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
coli
Klebsiella 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)