UknownIDProjectSummer2021.docx

UNKNOWN IDENTIFICATION

This exercise requires that you apply knowledge gained from your laboratory exercises. It will be necessary for you to complete each of the assignments yourself. The protocol and purpose of the various biochemical tests which you might find useful in your identification is located at the end of this document.

You will be issued an overnight culture of unknown bacteria. The culture will be tagged with a number. It is your responsibility to record that number for your reference and on every page of the report you hand to the instructor. Make observations of the broth culture as soon as you receive it using scientifically accepted terminology. Once that culture is transferred to your hands, it is your responsibility to maintain that culture in useable form and contaminant free!

Using aseptic technique, transfer a sample of the broth culture to two TSA (Tryptic Soy Agar) plates immediately and incubate the plates at 25 and 37oC to determine the appropriate growth temperature for the bacterium. Also inoculate one TSA slant and grow at 37oC as a backup in case your plates do not grow for some unpredictable reason. Remember to label every culture you create in the proper manner and discard all cultures appropriately when you are through with them.

Staining tests can be carried out on the broth culture on the day of issue prior to discarding the tube. It will be in your best interest to set up at least two Gram stains and one each of the following: acid-fast stain, endospore stain and negative stain. It is critical that you also perform the appropriate staining techniques on any colony or culture which you generate from the original culture to insure it matches the original stains.

Any subculture you create, must be monitored regularly and the appropriate observations concerning bacterial growth should be recorded in good scientific terminology. If your cultures become contaminated, it is your responsibility to rescue the appropriate organism.

Once you are sure you have established two pure agar slant cultures, you will use one as your working culture and the other as an emergency backup. Use only the working culture for biochemical tests or any other means of identification. Be sure to stain your organism after every biochemical test you perform. You may make a fresh subculture from the working stock whenever necessary. Before submitting your results, go back and insure by staining that your subcultures are identical to the emergency stock which was not handled.

PROCEDURE:

After colony characteristics, Gram stain reaction, and cell morphology have been determined and recorded locate the correct table for the unknown bacterium. Begin the identification process by performing the standard biochemical tests listed in the table. Instead of running every test on the table try to follow some systematic approach. While running one or more tests, begin planning what test (or tests) should logically follow either a positive or negative result. Continue this process until all the listed bacteria species are eliminated with the exception of one. This should be the identification of the unknown bacterium. Run two more test, the confirmatory tests, to verify the identification is correct.

If necessary, you may request specialized media or additional material if you can justify their uses and if we have them available in the laboratory. You may use any reference material you deem appropriate. Computer software in the Biology Study Center may help you. You are not to take your cultures out of the laboratory at any time.

Growth Characteristics of Unknowns (Figure 1):

Optimal Growth Temperature: Determine the optimum growth temperature by growing the unknown at 25oC and 37oC and note where the organism grows best. Most of the possible unknown organisms will be mesophilic and will grow well at either temperature. If an organism which is red at 25oC but only slightly pink at 37oC, it is a mistake to presume the organism prefers room temperature growth. 37oC is the optimum temperature of growth for Serratia marcescens. A characteristic phenotype such as a color is different from a growth rate. Preferred growth temperature will be established on the first streak plates generated from the unknown broth.

Colony Appearance: Record the growth characteristics of the bacterial colonies. Include descriptions of colony margins, colony diameter, elevation, color, consistency (Is it dry, mucoid, etc.?), degree of spreading. The following are terms used to describe colony characteristics on agar as you would see them with the naked eye. These terms must only be applied to isolated colonies not growth on slants or as a lawn. Colony form refers to the overall shape of the entire colony. Elevation refers to shape and distance a colony comes up from the surface of the agar while looking at the side of the colony. The margin refers to the shape of the colony b .

Staining Characteristics: Perform a Gram stain on the sample you obtain as your unknown. Every time you perform a test, you will want to insure that there is only one organism in the tube and that it is the correct organism. One wrong result can lead you on a wild goose chase and contamination will happen even to the most careful person when you least expect it. When you report your Gram staining results, they should include the Gram reaction as well as the morphology and any possible arrangements. For instance you would report that you have a Gram Negative Bacillus or a Gram Positive Coccus.

Growth Tests: You will also want to perform Gram Stains on cocci which have been grown in broth and compare them to stains from agar cultures. This is because all the cocci will have a staphylococcus arrangement when grown on agar. This is often an artificial result because you are scraping many groups off the agar and placing them on a slide where they are all bunched together. In a broth, you will see the true arrangement because the groupings space themselves out within the broth.

Motility testing: Use a 24-hour culture of the unknown to determine motility. It is best to wait until you have a pure culture of each unknown organism prior to performing motility testing using semisolid agar media. If you have a coccus, it will be nonmotile. It is a waste of your time and the lab media to perform motility testing on a coccus.

Oxygen Requirements: If you suspect that you have an anaerobic or facultatively anaerobic organism, you may want to grow the organism in the absence of oxygen. Growth of microaerophiles will be boosted if you use a candle jar; however, microaerophiles will not grow in an anaerobic chamber. Microaerophiles will grow as tiny, pinpoint, translucent colonies on agar in the presence of oxygen. Anaerobes, aerotolerant anaerobes and facultative anaerobes will grow in a GasPak canister. Additionally, using these techniques, you will be able to exclude aerobes.

Biochemical Tests

Sugar Fermentation Tests: Fermentation reactions can be tested on a variety of sugars (glucose, sucrose, lactose, sorbitol, mannitol, dulcitol, etc.). The test is performed using a broth with the appropriate sugar plus a phenol red indicator. The tubes are inoculated and incubated 24-72 hours at the optimum temperature. Acid production is indicated by a bright yellow color change. Gas production is seen as a gas bubble trapped inside the small inner tube (Durham tube). Some bacteria may grow in the broth without producing acid and gas and are scored as negative. Some bacteria will produce an alkaline pH which is seen as a pink or mauve color in the broth. In addition, contamination can result in an incorrect result. Perform Gram or other appropriate stains following a fermentation test to ensure that there is only one organism in the tube.

Gelatin Hydrolysis: The production of proteases may lead to gelatin hydrolysis. This test is performed in nutrient gelatin by inoculating the gelatin by stabbing and allowing it to incubate at the organism’s preferred temperature for 7-10 days. A positive result is determined when the solid gel is liquefied and remains liquid after the tube has been chilled to below 25oC. The tube is usually refrigerated 15-30 minutes (4oC) prior to reading results. Stain culture before placing in the refrigerator to confirm only one organism is growing in the tube. A positive gelatin hydrolysis test means the organism produces peptidases or proteases which break down protein in the medium and allow the bacterium to transport amino acids and small peptides into the cell for energy or protein synthesis. A positive test also may mean the organism is capable of spreading through tissue during infection by producing enzymes to degrade protein.

Fat Hydrolysis: The production of lipases results in breakdown of triglyceride (simple fat) into glycerol and three fatty acids. This test is performed by spot inoculating one unknown organism per plate in the center of a Spirit Blue Agar plate. A positive reaction is indicated by a lightening or clearing of the blue color. The color change is the result of the release of fatty acids which acidify the medium. The pH indicator in the agar responds to the drop of pH by changing to a lighter color. The plate should be incubated at the organism’s optimum temperature for 24-48 hours. Stain culture following reading.

Starch Hydrolysis: The production of amylase results in the breakdown of starch. Starch agar comes in the form of a deep and will need to be melted and poured into a petri plate. Spot inoculate the unknown onto starch agar plate and incubate at the optimum temperature for 24 hours. Prepare a smear for staining prior to adding the iodine. Flood the plate with Gram’s iodine and pour off residual iodine into a container of disinfectant. The iodine will stain intact starch dark brown or purple. The areas where the starch has been broken down by amylase will be clear because simple sugars do not stain.

Indole test: Some bacteria will hydrolyze tryptophan producing pyruvate, ammonia and indole. This test is performed by stab inoculating a SIM deep and incubating 24-48 hours at the optimum temperature. Prepare a smear from the growth at the top of the SIM agar prior to adding the Kovac’s reagent. 5 drops of Kovac’s reagent (dimethylaminobenzaldehyde) are added to the tube following growth. A positive test is read as a red ring at the top of the tube.

MRVP (Methyl Red/ Voges-Proskauer Test): Some strains of bacteria will produce a mixture of acids during fermentation which may be detected by the methyl red (MR) test. The test is performed by inoculating MRVP broth with the unknown and growing 24 hours at the optimum temperature. Prepare a smear of the growth before going on. Five drops of methyl red are added to the tube which is left undisturbed. A positive test results in a pink/red color of the medium. Positive MR indicates the presence of a mixed acid fermenter which converts glucose in MRVP broth to a mixture of acids and thus create a very low pH which is indicated by the red color of the Methyl Red pH indicator. The Voges-Proskauer (VP) test allows identification of butanediol fermenters by reaction with the neutral product acetoin. Butanediol fermenters produce acetoin as a neutral precursor produce of glucose fermentation before converting it to 2,3-butanediol and some acids as well. Because of the production of this alcohol and less acid, the pH of the medium is higher in this case. The unknown is added to MRVP broth and incubated at optimum temperature for 24 hours. 15 Drops of V-P reagent I and 5 drops of V-P reagent II are added to the tube. The tube is shaken to oxygenate the media and allowed to stand 15 minutes. A positive test is seen as a red to pink color at the surface of the liquid. A negative VP test is seen as a cloudy beige layer or no change.

Catalase Production: Tests for the production of the enzyme, catalase. Grow organism on TSA agar at the optimum temperature and prepare smear prior to testing. A drop of 3% hydrogen peroxide is placed on a bacterial colony. Production of bubbles is a positive test for the breakdown of hydrogen peroxide producing water and oxygen. This test cannot be performed on a blood agar surface; however, cells from a blood culture can be removed from the blood then tested.

Triple Sugar Iron Agar (TSI): TSI medium contains three sugars (glucose, sucrose and lactose) and iron salts. The test may be used to detect acid and gas production and also production of hydrogen sulfide. The slant is inoculated with the unknown by streaking the slant and then using the loop to stab through the agar. The tube is incubated for 24-72 hours and observed for the following reactions: Acid production = yellow color; Alkalinity = red color; Gas = bubbles or cracks in the agar; Hydrogen sulfide = blackening of the agar. Prepare a stain of growth seen.

Citrate Utilization: The unknown is streaked/stabbed into a Simmon’s citrate agar slant and allowed to grow at the optimum temperature. This test requires oxygen (loose cap), and the enzyme, citrate permease, must be present in the bacterium. If citrate is used as a carbon source, the agar will change color from green to royal blue. Stain organisms growing on agar. The color change is caused by the release of sodium carbonate (a base) which causes the bromothymol blue to turn from green to blue.

Litmus Milk Reactions: Litmus milk (containing 10% skim milk and litmus pH indicator) is inoculated with the unknown and allowed to incubate 2-7 days at the optimum temperature. There are several possible reactions. Pink color = fermentation of sugars with acid production; Purple = alkaline reaction; Peptonization = clearing of liquid indicates fat and protein hydrolysis; Reduction = a bone white color in the milk especially in the lower half; Coagulation = curd formation due to denaturation of milk proteins; Ropiness = trailing of strings or ropes of thickened material behind an inoculating loop when passed through the milk. Gas production will only be detected when coagulation takes place and is seen as cracks in the curd. Don’t forget to stain growth; however, be aware that a pink background color will develop when milk is Gram stained. The background may make it harder to verify Gram negative cells.

Mannitol Salt Agar (MSA): Used to distinguish the Gram-positive cocci from other organisms as well as other salt tolerant species. The agar contains mannitol (a sugar/alcohol), phenol red pH indicator and 5% sodium chloride (the Staphylococcus species are salt tolerant). The agar comes in the form of a deep. It may be stabbed and incubated. It may also be melted, poured into a plate and streaked (4 quads) when solid. Temperature of incubation depends on the organism’s optimum growth temperature. A positive MSA test is scored by growth and a bright yellow color change in the agar. The yellow color comes from the production of acid from fermentation of mannitol, and the phenol red pH indicator turns yellow in acid. Likewise, alkaline products cause phenol red to turn fuchsia. Some organisms will be salt tolerant, but will produce alkaline produces. These are scored as negative in the MSA test. Don’t be fooled if you have a yellow colored organism growing on the agar. Streptococcus and Enterococcus species grow more slowly on MSA with only slight fermentation. Micrococcus species grow slowest and are usually negative for the color change. Stain organisms following growth.

Oxidase Production: Tests for the production of the enzyme, oxidase, which is produced by certain bacteria. Grow bacteria on a TSA agar plate and prepare a smear prior to testing for oxidase. Temperature of incubation depends on the organism’s optimum growth temperature. Following growth, prepare a Gram of AF stain. Do not add chemical until staining result have been viewed and recorded. A drop of oxidase reagent (diphenylamine) is placed on a bacterial colony. The colony will darken to purple after 20-30 seconds if the test is positive. Any purple color after 30 seconds is read as a negative test because of exposure of the chemical to oxygen. Make sure the chemical is fresh before using.

Nitrate Reduction: Nitrate may be converted to nitrite by certain bacteria. The unknown is inoculated into nitrate broth and incubated at optimum temperature for 24-48 hours and a smear is prepared prior to going on. Be sure tube in incubated with tight cap since this test is used as in indicator of anaerobic respiration where the nitrate will be used as a final electron acceptor (therefore reduced). Also be sure to check for turbidity before performing a test. You will get false negative results if your organism does not grow. Equal volumes (5 drops) of reagent A (sulfanilic acid) and reagent B (napthylamine) are added to the tube. A positive test results in bright pink or blood red color development. Many Bacillus, Pseudomonas and Micrococcus species reduce nitrate. If no color develops, zinc is added to the tube. If the reaction was truly negative, nitrate will be reduced to nitrite, and the broth will turn red in the presence of zinc (negative test). If nitrite was reduced further to nitrogen or ammonia, zinc addition will still result in lack of color (this is a positive test) for nitrate reduction. It may take about five minutes for zinc to form a red color.

Eosin Methylene Blue (EMB) Agar: This medium is selective and differential for Gram negative bacteria because the dyes inhibit everything else. The dyes are also absorbed differently by each genus that tolerates them. The agar is dark purple when solid and is in the form of a deep. The agar must be melted and poured into a petri plate. When solid, perform a 4-quadrant streak. Grow 24-48 hours. Temperature of incubation depends on the organism’s optimum growth temperature. The colony color is distinctive for several genera: Escherichia = metallic green colonies; Enterobacter = mucoid colonies with dark centers (Fisheye); Proteus = thin, purple, flat, spreading colonies; Serratia = dark purple colonies, Pseudomonas and Alcaligenes = lavender colonies. Don’t forget to stain the organism following growth.

MacConkey Agar: This medium is selective and differential for enteric bacteria. The agar is light sensitive and is kept in a cabinet in the biology stock room. You must request it, then melt the agar and pour a petri plate. When solid, perform a 4-quadrant streak and grow 24-48 hours. Temperature of incubation depends on the organism’s optimum growth temperature. Bile salts in the medium inhibit growth of many bacteria especially Gram positives. Lactose fermenters appear as pink colonies. Non-lactose fermenters are white. Gram stain following growth.

Blood Agar (Hemolysis): 5% sheep erythrocytes in an agar base are used to determine if bacteria are capable of breaking down the red blood cells resulting in a clearing or other change in the agar. This is an important differentiating test for streptococci and staphylococci; however, this medium is expensive and should not be used unless truly warranted. For example, if you check the unknown charts, you should notice that all Gram-negative rods and Gram-positive rods, respectively, yield the same results on blood agar. The hemolysis test does not provide any relevant information toward the identification of organisms in the Gram negative or Gram-positive rod categories and should not be used. If plate is not expired, warm to room temperature. Streak the blood agar plate using a 4-quadrant streak and incubate for 24 hours. Check if results are unclear, recheck in 24 hours. Do not incubate longer than 48 hours total. Temperature of incubation depends on the organism’s optimum growth temperature. Beta hemolysis is indicated by complete clearing of the blood agar adjacent to the bacterial colony which means the RBCs were completely destroyed. Alpha hemolysis is usually seen as a greenish opaque color around the colony when you hold the plate up to the light indicating that the hemoglobin was modified into methemoglobin thus changing the color. Gamma hemolysis is inapparent hemolysis. However, if you scrape a colony off the plate, you may be able to see a clearing under the colony. Stain any growth seen to ensure no contamination has occurred.

Growth on a Slant

Figure 1: Colony morphology and growth characteristics of organisms on solid media.

Table 1:

Gram-negative Microorganisms

Cell Morphology = Rod (Bacillus)

TEST

MICROORGANISM & RESULTS

Alcaligenes

faecalis

Enterobacter

aerogenes

Escherichia

coli

Proteus

mirabilis

Proteus

vulgaris

Pseudomonas

aeruginosa

Carbohydrate Dulcitol

alkaline/
no gas

no reaction

weak acid

no reaction

no reaction

alkaline/
no gas

Carbohydrate
Glucose

alkaline/
no gas

acid/gas

acid/gas

acid/gas

acid/gas

alkaline/
no gas

Carbohydrate Lactose

alkaline/
no gas

acid/gas

acid/gas

no reaction

no reaction

alkaline/
no gas

Carbohydrate

Maltose

alkaline/
no gas

acid/gas

acid/gas

no reaction

no reaction

alkaline/
no gas

Carbohydrate

Manitol

alkaline/
no gas

acid/gas

acid/gas

no reaction

no reaction

alkaline/
no gas

Carbohydrate

Sorbitol

alkaline/
no gas

acid/gas

acid/gas

no reaction

no reaction

alkaline/
no gas

Carbohydrate

Sucrose

alkaline/
no gas

acid/gas

alkaline/no gas

alkaline/
no gas

alkaline/
no gas

alkaline/
no gas

Catalase

positive

positive

positive

positive

positive

positive

Citrate

positive

positive

negative

positive

positive

positive

Colony Appearance

On TSA Agar Plates

mucoid, spreading,
beige-pink

heavily mucoid,
round, beige, convex

mucoid, beige, spreading

swarming at
25 and 37 C, darker tan

swarming at
25 and 37 C,
darker tan

tan, circular, diffusible fluorescent green pigment

DNase

negative

negative

negative

negative

negative

negative

EMB

colorless

fisheye

metallic green

colorless

colorless

colorless

Gelatin

negative

negative

negative

positive

positive

positive

Hemolysis

gamma

gamma

gamma

gamma

gamma

beta

Indole

negative

negative

positive

negative

positive

negative

Lipid

positive

positive

positive

positive

positive

positive

TEST

MICROORGANISM & RESULTS (continued)

Acaligenes

faecalis

Enterobacter

aerogenes

Escherichia

coli

Proteus

mirabilis

Proteus

vulgaris

Pseudomonas

aeruginosa

Litmus Milk

Acid

negative

positive

positive

positive (weak)

positive (weak)

negative

Litmus Milk

Alkaline

negative

negative

negative

negative

negative

positive

Litmus Milk

Coagulation

negative

negative

positive

negative

negative

negative

Litmus Milk

Peptonization

negative

negative

negative

negative

negative

negative

Litmus Milk Reduction

negative

negative

negative

negative

negative

negative

Methyl Red

negative

negative

positive

positive

positive

negative

Motility

positive

positive

positive

positive

positive

positive

MSA

not tested

not tested

not tested

not tested

not tested

not tested

Nitrate

Reduction

negative w/Zi

positive

positive

positive

positive

positive w/Zi

Oxidase

positive

negative

negative

negative

negative

variable

Oxygen Preference

aerobe

facultative
anaerobe

facultative
anaerobe

facultative
anaerobe

facultative
anaerobe

aerobe

Starch

negative

negative

negative

negative

negative

negative

Temperature

Preference

37 C

37 C

37 C

37 C

37 C

37 C

TSI

Butt

alkaline/
no gas

acid/gas

acid/gas

acid/gas

acid/gas

no reaction

TSI

H2S

negative

negative

negative

positive

positive

negative

TSI

Slant

alkaline

acid

acid

alkaline

alkaline

alkaline

Urease

negative

negative

negative

positive

positive

positive

Voges – Proskauer

negative

positive

negative

negative

negative

negative

Table 2:

Gram-positive Microorganisms

Cell Morphology = (Rod) Bacillus

TEST

MICROORGANISM & RESULTS

Bacillus

cereus

Bacillus

subtilis

Bacillus

megaterium

Bacillus

mycoides

Corynebacterium

pseudodiphtheriticum

Lactobacillus

casei

Carbohydrate Dulcitol

not tested

not tested

not tested

not tested

no
reaction

not tested

Carbohydrate
Glucose

acid/
no gas

acid/
no gas

acid/
no gas

negative

peach-colored
result

acid/
no gas

Carbohydrate Lactose

no reaction

No reaction

no
reaction

no reaction

no
reaction

variable

Carbohydrate

Maltose

not tested

not tested

not tested

not tested

no
reaction

not tested

Carbohydrate

Manitol

not tested

not tested

not tested

not tested

no
reaction

not tested

Carbohydrate

Sorbitol

not tested

not tested

not tested

not tested

no
reaction

not tested

Carbohydrate Sucrose

acid/
no gas

acid/
no gas

acid/
no gas

no reaction

no
reaction

acid/
no gas

Catalase

positive

positive

positive

positive

negative

negative

Citrate

positive

positive

negative

negative

negative

negative

Colony Appearance

On TSA Agar Plates

large,
spreading,
waxy, flat

Large, creamy, spreading

dry, creamy
undulate
edges,
spreading

large,
spreading,
lacy edges

pleomorphic rods and
“” shapes,
partially acid fast,
translucent, small

small,
translucent,
glossy

DNase

positive

negative

positive

negative

positive

negative

EMB

not tested

not tested

not tested

not tested

not tested

not tested

Gelatin

positive

positive

positive

positive

negative

negative

Hemolysis

beta

beta

beta

beta

gamma

gamma

Indole

negative

negative

negative

negative

negative

negative

Lipid

positive

positive

positive

positive

positive

negative

TEST

MICROORGANISM & RESULTS (continued)

Bacillus

cereus

Bacillus

subtilis

Bacillus

megaterium

Bacillus

mycoides

Corynebacterium

pseudodiphtheriticum

Lactobacillus

casei

Litmus Milk

Acid

negative

negative

positive

negative

negative

negative

Litmus Milk

Alkaline

positive

positive

positive

negative

negative

positive

Litmus Milk

Coagulation

negative

negative

negative

negative

negative

positive

Litmus Milk

Peptonization

negative

positive

negative

positive

negative

positive

Litmus Milk Reduction

positive

positive

positive

negative

negative

positive

Methyl Red

negative

negative

positive

negative

negative

positive

Motility

negative

negative

positive

negative

negative

negative

MSA

positive

positive

alkaline

negative

negative

not tested

Nitrate

Reduction

positive

positive

positive

negative

negative

negative

Oxidase

negative

variable

variable

negative

negative

negative

Oxygen

Preference

facultative
anaerobe

aerobic

aerobe

facultative
anaerobe

facultative
anaerobe

facultative
anaerobe

Starch

positive

positive

positive

positive

negative

negative

Temperature

Preference

37 C

37 C

37 C

37 C

30 C

37 C

TSI
Butt

acid/
no gas

acid

acid/
no gas

no reaction

acid/
no gas

acid/
no gas

TSI

H2S

negative

negative

negative

negative

negative

negative

TSI

Slant

alkaline

alkaline

alkaline

acid

acid

acid

Urease

negative

negative

negative

positive

negative

negative

Voges – Proskauer

negative

negative

negative

negative

negative

negative

Table 3:

Gram-positive Microorganisms

Cell Morphology = Coccus

TEST

MICROORGANISM & RESULTS

Enterococcus

faecalis

Staphylococcus

aureus

Staphylococcus

epidermidis

Streptococcus lactis

Micrococcus luteus

Carbohydrate
Glucose

acid/
no gas

acid/
no gas

acid/
no gas

acid

negative

Carbohydrate Lactose

acid/
no gas

acid/
no gas

acid/
no gas

acid

negative

Carbohydrate Sucrose

acid/
no gas

acid/
no gas

acid/
no gas

acid

negative

Catalase

negative

positive

positive

negative

positive

Colony Appearance

On TSA Agar Plates

small, slow, translucent

Circular/beige or gold

circular,
stark white,

small, slow,
translucent

Convex, citron yellow

DNase

negative

positive

negative

negative

negative

Hemolysis

beta

beta

alpha

gamma

gamma

Lipid

negative

negative

negative

negative

negative

Litmus Milk

Acid

negative

negative

negative

Top positive

negative

Litmus Milk

Alkaline

positive

negative

negative

negative

negative

Litmus Milk

Coagulation

top positive

negative

negative

positive

negative

Litmus Milk

Peptonization

positive

negative

negative

top positive

negative

Litmus Milk Reduction

top positive

negative

negative

positive

negative

TEST

Enterococcus

faecalis

Staphylococcus aureus

Staphylococcus

epidermidis

Streptococcus

lactis

Micrococcus luteus

Methyl Red

positive

positive

positive

positive

negative

MSA

no reaction

Positive/acid

no reaction

acid

positive

Nitrate

Reduction

negative

positive

positive

negative

negative

Oxidase

negative

negative

negative

negative

positive

Oxygen

Preference

facultative
anaerobe

facultative
anaerobe

facultative
anaerobe

Microaerophilic/slightly facultative

aerobic

Temperature

Preference

37 C

37 C

37 C

37 C

30oC

TSI

Butt

acid/
no gas

acid

acid/
no gas

acid

negative

TSI

H2S

negative

negative

negative

negative

negative

TSI

Slant

acid

acid

acid

acid

negative

Urease

negative

negative

negative

negative

positive

Voges –

Proskauer

negative

negative

negative

negative

negative