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Home: CDC Streptococcus Lab > Protocols > Identification of other Streptococcus Species > Section 1

Identification of other Streptococcus Species: Streptococcus General Methods

Section 1

OUTLINE OF THE WORK FLOW FOR THE STRETOCOCCUS LABORATORY

  1. If the culture is an unidentified gram-positive coccus, an Enterococcus, viridans Streptococcus, or of unknown identity (basically includes all cultures other than pneumococci, ß-hemolytic streptococci, and nutritionally variant streptococci), inoculate the following media. Inoculate a trypticase soy 5% sheep blood agar plate by streaking a heavy inoculum onto one-fourth of the plate and streak the remaining portion for isolated colonies. Place a vancomycin disk on the heaviest part of the inoculum, and put the plate into a candle extinction jar or a CO2 incubator for 18 to 24 h at 35C.
  2. If the culture is identified as a beta-hemolytic streptococcus, or group A, B, C, F, or G streptococci, inoculate a trypticase soy 5% sheep blood agar plate and place a bacitracin disk on the heavy part of the growth. All plates should be incubated in a candle extinction jar or CO2 incubator for 18 h at 35ΕC. For most cultures submitted as group A streptococci, those that appear pure on the submitted culture, a 30 ml and a 5 ml Todd-Hewitt broth (THB) should be inoculated. Place the 5 ml THB in a 30Ε C incubator or leave at room temperature for 1-3 days. The 5 ml THB is used for T-agglutination typing of group A streptococci. Place the 30 ml broth at 35ΕC for 16 to 18 h. Do not incubate longer than 18 h.

    For beta-hemolytic streptococci other than group A, a 30 ml THB can also be inoculated and placed at 35ΕC for 18 to 24 h. Some strains may take more than one day of incubation, no harm is done by incubating these broths longer than 24 to 72 h. The 30 ml THB is used for serogrouping and serotyping in some cases.
  3. If the culture is submitted as a nutritionally deficient Streptococcus (NVS), inoculate an entire trypticase soy blood agar plate with the culture. Perpendicular to this streak, carefully make a single streak with a Staphylococcus aureus culture. This test will determine if the unknown culture forms satellite colonies adjacent to the staphylococci, a characteristic of all NVS. Incubate the plate in CO2 or a candle extinction jar at 35Ε C for 24 to 48 h.

Examples of unusual or unexpected results:

The blood agar plates used in the manner described above are for checking for purity of cultures. If any of the results are unusual or not expected or the culture is contaminated the test must be repeated.

Vancomycin resistant streptococci or other unknowns other than leuconostocs and pediococci, which are intrinsically vancomycin resistant.

AccuProbe-Enterococcus Test

 

I. Principle
The AccuProbe-Enterococcus test is used to aid in the identification of atypical enterococci and to help differentiate between Enterococcus and Lactococcus strains.
II. Inoculum
An overnight culture grown on blood agar incubated 35°C in CO2.
III. Reagents and Materials
Genprobe Accuprobe Enterococcus Culture Identification Test, GEN-PROBE Inc. San Diego, CA
IV. Procedure
The test is performed according to the package insert instructions.
V Reading and Interpretation
Automated
VI Limitations
Use care with the amount of colonies used. Too many colonies will result in a false positive test.
VII

Quality Control
Quality controls, positive and negative reactions are determined each day the test is determined. E. faecalis SS1273 and S. sanguinis SS910 are used as the positive and negative controls respectively.

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AccuProbe-Pneumococcus Test

I. Principle
The AccuProbe-Pneumococcus test is used to aid in the identification of atypical pneumococci and to help differentiate between viridans Streptococcus strains.
II. Inoculum
An overnight culture grown on blood agar incubated 35°C in CO2.
III. Reagents and Materials
Genprobe Accuprobe Pneumococcus Culture Identification Test, GEN-PROBE Inc. San Diego, CA
IV. Procedure
The test is performed according to the package insert instructions.
V Reading and Interpretation
Automated
VI Limitations
Use care with the amount of colonies used. Too many colonies will result in a false positive test.
VII

Quality Control
Quality controls, positive and negative reactions are determined each day the test is determined. S. pneumonia and S. sanguinis SS910 are used as the positive and negative controls respectively.

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Acid Formation in Carbohydrate Broths

I. Principle
The ability of bacteria to form acid in some carbohydrate broths and not in others can be used in identification schemes. If the bacteria acidify the carbohydrate, the pH will change and the indicator (brom cresol purple) will turn yellow.
II. Inoculum
An overnight culture in Todd Hewitt broth incubated over night at 35° C or a fresh bacterial suspension in Todd Hewitt broth may be used as the inoculum.
III.

Reagents and Materials

1. Heart Infusion broth with 1% carbohydrate and 0.16 brom cresol purple indicator. Most of the carbohydrate broths are commercially available (Remel). An asterisk indicates those that are made by CDC media lab.
2. Pipet

 
IV.

Procedure

1. Inoculate carbohydrate broth tube with 1-3 drops of inoculum. 2. The broth tube is then incubated at 35ΕC for up to 7 days in ambient air. Fastidious organisms may be held up to 14d.

 
V Reading and Interpretation
A positive reaction is recorded when the broth turns yellow. A negative reaction is when no color change occurs. A definite color change that is not quite yellow may be interpreted as a weak positive reaction.
VI Limitations
Do not incubate in CO2 as this may alter the pH.
VII Quality Control
Each lot and shipment of carbohydrate broth medium is tested for positive and negative reactions upon receipt in the laboratory. The strains and reactions for each broth are listed below.

 

 

Carbohydrate
Positive Reaction
Strain # species
Negative Reaction
Strain # species
Arabinose SS-1274, E. faecium SS-1273, E. faecalis
Glycerol SS-1273, E. faecalis SS-2174, E. faecium
Inulin SS-1229, E. casseliflavus SS-429. S. mitis
Lactose SS-1273, E. faecalis SS-1419, P. acidilactici
Lactose SS-1273, E. faecalis SS-1419, P. acidilactici
Maltose SS-1273, E. faecalis SS-1419, P. acidilactici
Mannitol SS-1273, E. faecalis SS-1419, P. acidilactici
Melebiose SS-1229, E. casseliflavus SS-1273, E. faecalis
*m-α-D-glucopyranoside SS-1229, E. casseliflavus SS-1273, E. faecalis
*Pullulan SS-1633, G. balaenoptera SS-1138, G. adiacens
Raffinose SS-1229, E. casseliflavus SS-1273, E. faecalis
Ribose SS-1273, E. faecalis SS-1317 E. casseliflavus
Sorbitol SS-1273, E. faecalis SS-1227, E. hirae
Sorbose SS-817, E. avium SS-1273, E. faecalis
Sucrose SS-1273, E. faecalis SS-1419, P. acidilactici
*Tagatose SS-1138, G. adiacens SS-1633, G. balaenoptera
Trehalose SS-1273, E. faecalis SS-1344, S. equi
Xylose SS-1503, E. porcinus

SS1404, E. ratti

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Arginine Hydrolysis

I. Principle
Certain bacteria contain the enzymes to hydrolyze arginine. This hydrolysis results in an alkaline change in the media results in a color change in the media. This test can be used for differentiated different bacteria.
II. Inoculum
A drop of Todd Hewitt broth culture grown overnight is the preferred inoculum. Alternatively a suspension in Todd Hewitt broth from growth on a plate or a tiny amount of growth from a plate may be used as the inoculum.
III. Reagents and Materials
1. Moeller’s decarboxylase broth containing arginine. The medium is commercially available. 2. Pipet or loop
IV. Procedure
1. Add 1-3 drops of culture suspension to the tube of Moeller's decarboxylase medium containing arginine
2. Immediately overlay with sterile mineral oil (about 1 to 2 ml).
3. The medium is incubated at 35Ε C for up to 7 days in ambient air. (Some fastidious organism may be held up to 14d.)
V Reading and Interpretation
A positive reaction is recorded with the broth turns a deep purple color indicating an alkaline reaction, NH3 is released. The development of a yellow color or no change in color of the broth indicates a negative reaction.
VI

Quality Control
Each new lot and shipment of medium is tested for positive and negative reactions. E. faecalis strain SS1273 is used for determining positive reactions and S. avium strain SS817 is used for determining negative reactions.

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Bacitracin Test

I. Principle
The bacitracin disk is sensitivity test used to differentiate the beta- hemolytic Streptococcus.
II. Inoculum
An overnight culture grown on 5% sheep blood agar incubated 35°C in CO2.
III. Reagents and Materials
1. bacitracin “A” disk (BBL)
IV.

Procedure

1. Select a beta-hemolytic colony and heavily inoculate a quadrant of a 5% sheep blood agar plate.
2. Drop an “A” disk in the heaviest zone of inoculation.
3. Tap disk lightly to ensure that it adheres to the agar.
4. Incubate plate overnight in CO2 at 35°C.
V Reading and Interpretation
Any zone of inhibition is considered a positive test or sensitive test. Growth to the edge of the disk is interpreted as a negative test or resistant test.
VI Limitations
VII

Quality Control
Quality Control is performed on each shipment and lot of bacitracin disk. Streptococcus pyogenes is the positive (sensitive control) and Enterococcus faecalis SS1273 is the resistant or negative control. Results are recorded in the QC log book.

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Bile Esculin Test

I. Principle
A selective and differential medium used in the identification of catalase-negative bacteria. The selective agent bile, inhibits most gram positive bacteria. The enterococci and Streptococcus bovis will grow. Esculin in the medium is hydrolyzed to esculetin and dextrose. The esculetin reacts with ferric chloride in the media to form a black-brown color.
II. Inoculum
An overnight culture in Todd Hewitt broth incubated over night at 35° C or a fresh bacterial suspension in Todd Hewitt broth may be used as the inoculum. An inoculating loopful of culture may also be used.
III. Reagents and Materials
1. Bile esculin slant (Remel)
IV. Procedure
1. Inoculate tube with 1 drop of inoculum allowing drop to run down slant. Alternatively, the slant may be inoculated with a loopful of growth from a blood agar plate.
2. The slant is then incubated at 35ΕC for 2 days in ambient air. Fastidious organisms may be held up to 14d.
V Reading and Interpretation
The bile esculin test is positive when a black color forms over one-half or more of the slant. If no blackening occurs the test is negative.
VI Limitations
Do not incubate medium in a carbon dioxide atmosphere. The increase in C02 will cause the viridans streptococci to grow better and increase the likelihood of a positive BE reaction. Streptococcus bovis and enterococci do not require C02 for good growth.
VII

Quality Control
Positive and negative reactions are determined on each new lot and shipment of media. Enterococcus faecalis strain SS-1273 is used for positive control reactions and Streptococcus sanguinis strain SS-910 is used for negative control reactions. Results are recorded in the QC log book.

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Bile Solubility Test

I. Principle
The purpose of the bile solubility test is to aid in the differentiation of S. pneumoniae from all other alpha-hemolytic streptococci. Sodium deoxycholate (2%) acts on the cell wall of pneumococci resulting in lysis.
II. Inoculum
An overnight culture grown on blood agar incubated 35°C in CO2.
III. Reagents and Materials
1. 2% deoxycholate (CDC Central Services Laboratory, formula #5333) 2. physiologic saline pH 7.0 3.13 X 100mm glass tube
IV. Procedure
1. Make a 1.0 ml saline suspension of cells from growth on an agar plate. A turbidity equal to that of 1.0 to 2.0 McFarland density standard should be used.
2. After a satisfactory density is achieved, divide the suspension into 2 tubes with approximately 0.5 ml in each.
3. Add 0.5 ml of 2% sodium deoxycholate (bile salts) to one tube and 0.5 ml saline to the other tube. Mix by vigorous shaking.
4. Incubate the tubes at 35-37Ε C for up to 2 h.
V Reading and Interpretation
Examine for clearing of the turbidity periodically. A clearing of the turbidity in the bile tube but not in the saline control tube indicates a positive test, i.e., the pneumococcal cells have lysed ("solubilized"). If the tube containing the cells and bile have not cleared the test is negative. On occasion some strains of pneumococci are only partially soluble in the bile salts, that is, a partial clearing occurs. These strains must have the proper zone of inhibition around the optochin test to be called pneumococci. Partially soluble strains with zones of inhibition of less than 14 mm are not considered pneumococci.
VI Limitations
The turbidity must be sufficient to detect a difference in the saline control tube.
VII

Quality Control
Each new lot of deoxycholate is tested for positive and negative reactions with S. pneumoniae strain ATCC-49619 (positive) and S. mitis strain SS-429 (negative). Results are recorded in QC log book.

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Camp Test

I. Principle
Some bacteria produce CAMP factor (a diffusible extracelluar protein) that synergistically acts with the beta-lysin of Staphylococcus aureus and enhances the lysis of red blood cells. The purpose of the CAMP test is to aid in the identification of nonhemolytic group B streptococci and other ß-hemolytic streptococci.
II. Inoculum
Growth from a blood agar plate or any solid media.
III. Reagents and Materials
TSA-sheep blood agar
IV. Procedure
1. The CAMP test is performed on TSA-sheep blood agar. A single streak of ß-lysin producing S. aureus made across the center of the plate. Strain SS-695 (Strep. Lab number is a ß-lysin producing strain of S. aureus.
2. A single colony of the unknown strain (beta hemolytic streptococci) is picked up with an inoculating loop and used to make a single streak perpendicular but not touching the S. aureus streak. A 2-3 mm space should remain between the streaks.
3. Incubate the inoculated plate normal atmosphere overnight at 35ƒ£C. Group B streptococci and a few other beta-streptococci produce an enhancement of the ß-lysin activity of the S. aureus strain.
V Reading and Interpretation
This enhanced activity is in the shape of an arrowhead at the juncture of the two streaks, with the widest portion of the arrowhead on the group B side.
VI Limitations
Do not incubate in an anaerobic environment or under CO2. Some S. pyogenes strains will give a positive reaction when incubated in CO2.
VII

Quality Control
Commercially available TSA-sheep blood agar does not always demonstrate the correct CAMP reaction. Therefore it is necessary to test a known group B streptococcus for CAMP reaction as a positive control on each test plate. Group B streptococcus strain SS-617 should be used as a positive control on each test plate.

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Catalase Test

I. Principle
Hydrogen peroxide is used (H2O2) to determine if bacteria produce the enzyme catalase.
II. Inoculum
Cultures that are grown on a blood free media or a colony grown on a blood agar plate that is carefully transferred to a slide without carry-over of any of the erythrocytes. Cultures are typically grown overnight at 35°C in CO2.
III. Reagents and Materials
1. Three percent hydrogen peroxide is obtained from a commercial drug store.
2. Pipet
3. Slides
IV. Procedure
1. The catalase test is best performed by flooding the growth of the bacteria (usually on an agar slant but blood free agar plates can be used) in question with 1.0 ml of 3% hydrogen peroxide and observing for effervescence (bubbling) which indicates a positive test. The bacteria must be grown on blood free medium.
2. Modifications of the catalase test may be performed by very carefully removing a colony of growth from a blood agar plate with a plastic needle or wooden applicator stick and transferring the colony to a glass slide. A drop of 3% hydrogen peroxide is added to the colony on the slide and observed for effervescence.
V Reading and Interpretation
Any sign of bubbling is interpreted as a positive test. The absence of bubbling is interpreted as negative.
VI Limitations
False positive results will result if any red bloods cell are transferred. Weak positive results should be repeated on a blood free medium. The catalase test gives the majority of differentiations very efficiently, however, there will be occasions when the catalase test and colony morphology will be misleading.
VII

Quality Control
The catalase quality control is performed once per lot and shipment. For positive reaction use a blood-free culture of Staphylococcus aureus: i. e., Cowen strain I but other confirmed Staphylococcal cultures can be used. For negative reaction use Streptococcus sanguinis strain SS-910 (ATCC-10556). Record in QC manual.

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Clindamycin Test

I. Principle
Resistance of bacteria to clindamycin is determined by using a clindamycin disk at a concentration of 2µg/ml. This resistance is useful in differentiating the Lactococcus species.
II. Inoculum
Growth from a blood agar plate or any solid media.
III. Reagents and Materials
1. clindamycin disk 2µg/ml 2. blood agar plate
IV. Procedure
Bacteria are spread onto a 5% sheep blood agar plate, the disk placed on the plate and the plate incubated at 35ΕC for 18-24 hr in CO2.
V Reading and Interpretation
Any zone of inhibition around the disk is considered sensitive. Zones are usually Ξ20mm.
VI Limitations
VII

Quality Control
The quality control is tested with each lot and shipment. Lactococcus lactis is used for the negative control (Sensitive) and L. garviae is used for the positive control (Resistant).

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Esculin Hydrolysis

I. Principle
A differential medium used in the identification of catalase-negative bacteria. Esculin in the medium is hydrolyzed to esculetin and dextrose. The esculetin reacts with ferric chloride in the media to form a black-brown color.
II. Inoculum
An overnight culture in Todd Hewitt broth incubated over night at 35° C or a fresh bacterial suspension in Todd Hewitt broth may be used as the inoculum. An inoculating loopful of culture from a blood agar plate may also be used.
III. Reagents and Materials
1. Esculin slant (Remel)
IV. Procedure
1. Inoculate slant tube with 1-3 drops of inoculum allowing drop to run down slant. Alternatively, the slant may be inoculated with a loopful of growth from a blood agar plate.
2. The slant is then incubated at 35ΕC for 7 days in ambient air. Fastidious organisms may be held up to 14d.
V Reading and Interpretation
The esculin test is positive when a black color forms over one-half or more of the slant. If no blackening occurs the test is negative.
VI Limitations
Do not incubate medium in a carbon dioxide atmosphere. The increase in C02 will cause the viridans streptococci to grow better and increase the likelihood of a positive reaction. Streptococcus bovis and enterococci do not require C02 for good growth.
VII

Quality Control
Positive and negative reactions are determined on each new lot and shipment of media. Enterococcus faecalis strain SS-1273 is used for positive control reactions and Streptococcus mitis strain SS-429 is used for negative control reactions. Results are recorded in the QC log book.

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Gas from MRS broth

I. Principle
The production of gas from glucose is tested in Lactobacillus MRS broth.
II. Inoculum
An overnight culture in Todd Hewitt broth incubated overnight at 35° C or a fresh bacterial suspension in Todd Hewitt broth may be used as the inoculum. An inoculating loopful of culture from a blood agar plate may also be used.
III. Reagents and Materials
The MRS broth is prepared in the CDC Central Services Laboratory, formula No. 9208. The petroleum jelly is also prepared in the CDC Central Services Laboratory, formula No. 9356.
IV. Procedure
The broth is inoculated with 2 or more colonies from a plate or with 1 to 2 drops of broth culture. The broth is then sealed with melted petroleum jelly and, the tube is incubated at ambient air 37° C up to 7 days.
V Reading and Interpretation
Gas production is indicated by the gas formation between the broth and the petroleum jelly plug which pushes the wax plug toward the top of the tube. Small bubbles that may accumulate over the incubation period are not read as positive, only when the wax plug is separated from the broth is the test read positive. Most leuconostoc strains are positive at 24 h but some strains may take longer.
VI Limitations
VII

Quality Control
Each new lot of MRS broth prepared by the CDC Central Services Laboratory is tested for positive (gas production) and negative (no gas production) reactions. Leuconostoc mesenteroides strain SS-1238 (ATCC-8293) is used for positive and Streptococcus sanguinis strain SS-910 (ATCC-10556) is used for negative gas production. Results are recorded in the QC log.

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Gram Stain

I. Principle
The gram stain is used to differentiate between gram-positive and gram-negative bacteria. Cellular morphology can also be determined. Gram-positive and gram-negative bacteria are both stained by crystal violet. The addition of iodine forms a complex within the cell wall. Addition of a decolorizer removes the stain from gram-negative organisms due to their increased lipid content. These cells are stained pink with the counter stain safranin.
II. Inoculum
The gram stain can be performed on the growth of any strain grown on any type of media. However, for this group of bacteria (gram-positive cocci), it is best performed on the growth of bacteria in thioglycolate broth at 24h incubation. The staining procedure is modified when preparing the smear from thioglycolate broth. The smear can not be fixed to the slide with hear but must be fixed with methanol.
III. Reagents and Materials
1. Crystal Violet Stain
2. Gram Iodine (Combine Gram Iodine Concentrate to Gram Iodine Diluent)
3. Decolorizer Solution
4. Methanol
5. Slides
6. Inoculating loop
7. Microscope with Immersion Objective
IV.

Procedure

1. Spread single loop of culture from the thioglycolate broth to a microscope slide. Spread the culture over 1/3 to 1/2 to the total area of the slide.
2. Allow the smear to air dry. This may take up to 1 hour depending on the temperature and humidity of the room.
3. Cover the entire bacterial smear with 3 or 4 drops of methanol to fix the smear and allow to air dry. Again this may take up to an hour.
4. Cover the bacterial smear with crystal violet stain and allow to stand 1 minute. Gently was the stain off with cool tap water and drain water from slide.
5. Cover the smear with grams iodine and allow to stand 1 minute. Gently wash the iodine off with water and drain the water from the slide.
6. Rinse the bacterial smear with decolorizer solution for 10 seconds; decolorization is complete when the solution runs clear from the slide. Gently rinse with water and drain the slide.
7. Cover the bacterial smear with safranin stain, and allow to stand for 1 minute, then gently wash the stain from the slide.
8. Blot the slide dry with absorbent paper and examine the slide under oil immersion lens.
V Reading and Interpretation
The gram stain is used to aid in the differentiation of the gram positive cocci. The arrangement of the cells is what helps to differentiate the genera. Bacteria that divide on random planes form grape-like clusters of cells. This is the type of arrangement commonly observed with staphylococci. Bacteria that divide on one plane form pairs and eventually form chains if the cells remain attached to each other. This type of cellular morphology is observed with streptococci. Bacteria that divide on two planes at right angles form packets of fours or tetrads. This type of arrangement is observed with the aerococci.

One of the most difficult tasks that microbiologist have is determining whether or not the cellular morphology of the cells are actually cocci or short rods. Since many of the lactobacilli are gram positive short rods in chains, they are sometimes confused with the streptococci. The clinical sources and colonial morphology on blood agar plates of the lactobacilli are also similar to the streptococci, especially members of the viridans streptococci. When reading the gram stain, remember that the cellular arrangement is never 100% chains, pairs, tetrads, or clusters. The microbiologist must determine the most common cellular arrangement. For example, for the Gemella species, one might observe some pairs and short chains as well as tetrads. If tetrads are observed in most fields under observation, then the strain is dividing on two planes and this should be recorded.
VI Limitations
Younger cultures give more characteristic observations that older ones. Older cultures may stain gram negative. Stains exposed to antimicrobial reagents may have atypical morphology and are more susceptible to decolorization. Gram positive organisms that are over-decolorized will appear gram-negative.
VII Quality Control
The gram stain quality control is performed once per week .InoculateStreptococcus sanguinis strain SS910 and Escherichia coli 25922 into thioglycolate broth medium and incubate overnight at 35° C ambient air. Prepare the slide using 1 loopful of each culture on the same slide. Slides may be fixed in advance and stored. The completed procedure should show gram-positive cocci in chains and gram-negative rods. Record results in QC manual.
VIII

References
Murray, P.R., Baron, E. J., Jorgensen, J.J., Pfaller, M.A., and Yolken, R.H. Manual of Clinical Microbiology, 8th ed. ASM Press: Washington, DC, 2003.

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Growth at 10C and 45C

I. Principle
Growth at 10C and 45C is determined in heart infusion broth base medium and can be used as differential test for catalase-negative gram positive cocci.
II. Inoculum
An overnight culture in Todd Hewitt broth incubated overnight at 35° C or a fresh bacterial suspension in Todd Hewitt broth may be used as the inoculum. An inoculating loopful of culture may also be used
III. Reagents and Materials
1. Heart infusion broth base medium Remel number 061030.
IV. Procedure
1. Two of the broth tubes are inoculated with one or two colonies or one to two drops of an overnight Todd Hewitt broth culture.
2. Incubate each tube at the respective temperatures, 10C and 45C. For the 10C incubator, a refrigerator that can be adjusted to hold a temperature of 10C is satisfactory. The refrigerator can be used for storage of media and other materials. For the 45C incubator, a hot water bath that is adjusted to hold a temperature at 45C is best.
3. The tests are held a minimum of 7 days and up to 14 days in the case of slow growing strains.
V Reading and Interpretation
An increase in turbidity indicates growth and a positive test. Color changes are not required for a positive test.
VI Limitations
Be sure that the water level in the 45C water bath is above the level of media in the tubes. In addition, the caps of the medium tubes should be carefully sealed with
VII

Quality Control
Each new lot of broth is tested for positive and negative reactions. Enterococcus faecalis strain SS-1273 is used for positive (growth) reactions at both 10C and 45C. Streptococcus sanguinis strain SS-910 is used for negative reactions (no growth) at both 10C and 45C.

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Hemolysis

I. Principle
The hemolytic reaction is particularly useful in the differentiation of the Streptococci. The hemolytic reaction is determined on agar media containing 5% animal blood. The most commonly used base medium is trypticase soy agar and the most commonly used blood is sheep blood. The reason for the use of trypticase soy base is that it supports the growth of all the bacteria listed in Table 2. Other base media may be substituted if control strains of all genera are tested for growth. Sheep blood is used because of the convenience in testing throat swabs for ß-hemolytic streptococci. Sheep blood does not support the growth of Haemophilus haemolyticus which appears similar to streptococci on agar containing rabbit, horse, or human blood.
II. Inoculum
Pure culture on solid media.
III. Reagents and Materials
1. Trypticase soy agar plates containing 5% sheep blood are obtained from Becton-Dickinson Microbiology Systems, Cockysville Md., product No. 21261.
IV. Procedure
1. Streak culture for isolation on TSA plate with 5% sheep blood.
2. Incubate plate at 35°C in CO2 for 24 h.
V Reading and Interpretation
A beta-hemolytic reaction is interpreted as complete clearing around the colony. An alpha-hemolyic reaction is interpreted as greening around the colony and gamma hemolysis is interpreted as no change in the media surrounding the colony. The hemolytic reaction on blood agar is complex and subject to many variables. For the complete explanation and interpretation of the reactions the reader is referred to the 1977 publication, CDC Laboratory Update, Isolation and Identification of Streptococci. Part I. Collection, Transport, and Determination Hemolysis, Annex 1. Application of the interpretations of the hemolytic reactions for streptococci described in the Update to the other genera should not be a problem.
VI Limitations
VII

Quality Control
Under normal operating conditions, the blood agar plates are not quality controlled in our laboratory but are quality controlled by Becton-Dickinson Microbiology Systems. The hemolytic reaction of most bacteria submitted to this laboratory is known and identified with the culture upon submission. If a problem should be identified then quality control strains are used for testing the proper hemolytic reactions. Streptococcus pyogenes strain SS-103 is used for determining beta-hemolysis, Streptococcus sanguinis strain SS-910 is used for determining alpha-hemolysis, and Streptococcus bovis strain SS-752 is used for determining no reaction (neither beta or alpha hemolysis) on trypticase soy 5% sheep blood agar plates. All reactions are determined according to the 1977 CDC Laboratory Update, Isolation and Identification of Streptococci. Part I. Collection, Transport and Determination of Hemolysis (See annex 1).

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Hipppurate Hydrolysis Test

I. Principle
Some bacteria produce the enzyme hippurate hydrolase which hydrolyzes sodium hippurate to form benzoic acid and glycine. The addition of ferric chloride to benzoic acid forms an insoluble brown ferric benzoate precipitate.
II. Inoculum
An overnight culture in Todd Hewitt broth incubated at 35° C or a fresh bacterial suspension in Todd Hewitt broth may be used as the inoculum. An inoculating loopful of culture from a blood agar plate may also be used.
III. Reagents and Materials
1. Hippurate broth commercial suppliers.
2. Ferric choride (FeCl3) commercial supplies. Labeled as TDA if purchased for bioMereiux
IV. Procedure
1. The hippurate broth is inoculated with one drop of a fresh (16-20 h) Todd-Hewitt broth culture.
2. The broth is incubated for up to 7 days or until turbid growth is seen at 35ΕC.
  The tube of broth is then centrifuged to sediment the bacteria.
  Pipette 0.8 ml the clear supernatant to a small clear tube (13 x 100).
  Add 0.2 ml of ferric chloride reagent to the supernatant. Mix well.
V Reading and Interpretaion
A heavy precipitate that does not clear within 10 minutes indicates a positive test. A clear golden-brown liquid indicates a negative test.
VI Limitations
Growth should be turbid before testing. Some fastidious organisms may show poor growth.
VII

Quality Control
Group B Streptococcus strain SS-620 is used as a positive control and E. avium strain SS-817 is used as a negative control. QC is performed with each new lot number and shipment of hippurate broth and with each new lot and shipment of ferric chloride reagent. The quality control test results are recorded in the QC log.

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Lancefield Group Antigen

I. Principle
The purpose of determining the group antigen of ß-hemolytic streptococci is identify the species or species/group of streptococci as originally described by Rebecca Lancefield. Acid extraction is used to remove the serogroup from the cell.
II. Inoculum
An overnight 50 ml culture in Todd Hewitt broth incubated at 35° C.
III.

Reagents and Materials

1. 50 ml culture
2. 50 µl capillary tube
3. m-cresol purple
4. 13 X 100 mm test tube
5. boiling water bath
6. centrifuge
7. 1 cc vial
8. Serogrouping Reagents

 
IV.

Procedure

1. Centrifuge cells in 20 minutes at 2000 rpm to achieve a cell pellet.
2. Aspirate off the supernatant.
3. Add 2-3 drops m-cresol purple. Add .1 N HCl dropwise until a color change to pink. Vortex.
4. Place tube in a boiling water bath for 10 minutes.
5. After allowing to cool, transfer contents to a 13 X100 test tube.
6. Centrifuge at 2800 rpm for 10 minutes to remove any precipitate.
7. Transfer to a clean 13 X 100 mm test tube
8. Add .5 N NaOH dropwise until a change to purple.
9. Centrifuge at 2800 rpm for 10 minutes to remove any precipitate.
10. Transfer to a 1ml screwcap storage vial.
11. Set of capillary tubes by adding approximately 1 cm of serogrouping reagent and 1cm of extract. Careful not to get any space or bubbles between the two!
12. Cap the end with clay and set in clay rack.
13. Observe for up to 30 minutes.
V Reading and Interpretaion
A definite line or zone of precipitation that forms between the two is considered positive.
VI Limitations
Group D sometimes give weak reactions.
VII

Quality Control
Quality control is performed with the preparations of each batch of serogrouping reagent.

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Leucine amino peptidase (LAP)

I. Principle
Some bacteria produce leucine aminopeptidase which hydrolyzes the substrate leucine-β-naphthylamide to form β-naphthylamine. A pink to red color forms when p-dimethylaminocinnamaldehyde (PYR reagent) is added to β-naphthylamine.
II. Inoculum
Strains are grown on blood agar plates overnight at 35°C in CO2 for most gram positive bacteria. More than 1 day of incubation may be necessary for more fastidious genera such as the gemellae, alloiococci, and helcococci. The strains to be tested are grown on a blood agar plate until sufficient growth is seen to heavily inoculate the disks.
III. Reagents and Materials
LAP disk (Remel)
PYR reagent
Loops
Deionized Sterile water
IV.

Procedure
The procedure that is used in the Streptococcus laboratory is modified from the package insert. The PYR test is usually done simultaneously.

1. Place the disks on blood agar plate in an area of little or no growth or on a slide. The moisture from the plate is usually sufficient to rehydrate the disk. If the disk is placed on a slide, then a tiny drop of sterile deionized water is added. (DO NOT OVERSATURATE THE DISK). It is convenient to place the LAP disk on the left (L=LAP).
2. Using a loop or wooden stick, inoculate the disks heavily. Using two or more loop-fulls of culture is necessary for satisfactory results.
3. Leave the plates with the disks on the bench at room temperature for 10 minutes.
4. Add the detection reagent and read after 3 minutes.

 
V Reading and Interpretaion
The development of a red color within 3 minutes is positive. No change in color or a yellow color is negative. The color develops immediately. Discard the test after 10 minutes.
VI Limitations
False negative reactions may result if too little inoculum is used.
VII

Quality Control
Each lot and shipment of LAP disks are tested for positive and negative reactions. Enterococcus faecalis strain SS-498 is used for positive reaction and Aerococcus viridans strain SS-1251 (ATCC-11563) is used for a negative reaction.

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Litmus Milk Test

I. Principle
The purpose of the litmus milk test is to determine the acidification and clot of the milk in this test. This tests aids in the differentiation of the Leuconostoc species.
II. Inoculum
An overnight culture in Todd Hewitt broth incubated at 35° C or a fresh bacterial suspension in Todd Hewitt broth may be used as the inoculum. An inoculating loopful of culture may also be used.
III. Reagents and Materials
Litmus milk is obtained from commercial suppliers using their quality control.
IV. Procedure
1. Tubes containing litmus milk are inoculated with one drop of an overnight Todd-Hewitt broth culture.
2. Incubate at 35ΕC for up to 7 days in ambient air. Some fastidious strains may be held for 14 d.
V Reading and Interpretaion
The tubes are inspected for color change. The tubes begin a light blue color. Acid formation is indicated by first a pink color and then changes to white on continued incubation. A negative reaction is indicated by no color change. The tubes are also examined for clot, or solidification of the tube contents. Partial or complete solidification of the tube contents indicates a positive reaction. A negative reaction is indicated by no change in the consistency of the tube contents.
VI Limitations
VII

Quality Control
Enterococcus faecalis SS-1273 is used as a positive control and E. hirae strain SS-1227 is used as a negative control. QC is performed with each new lot number and shipment. The quality control test results are recorded in the QC manual.

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Motility Test

I. Principle
The ability of bacteria to move through a semisolid media is useful in differentiating bacteria. This test is particularly useful in differentiating the enterococci.
II. Inoculum
Strains are grown on blood agar plates overnight at 35°C in CO2 for most gram positive bacteria. More than 1 day of incubation may be necessary for more fastidious genera such as the gemellae, alloiococci, and helcococci.
III.

Reagents and Materials

1. motility test medium (Remel, number 061408)
2. inoculating needle
IV. Procedure
1. The medium is inoculated with an inoculating needle, not a loop. Apply a colony to the end of the needle from the agar plate.
2. The needle is inserted into the center of the medium in the tube for about one inch.
3. The inoculated tube is incubated at 30C in ambient air and incubated until good growth is observed, in most cases 24 to 48 h is sufficient.
V Reading and Interpretaion
Strains that are motile will show growth outward to the edge of the tube and downward toward the bottom of the tube. Negative strains will only show growth in the line of the stab.
VI Limitations
Do not place the motility test at 37C. Some strains become nonmotile at 37C but are motile at temperatures 25C to 30C.
VII

Quality Control
Enterococcus gallinarium SS-1228 is used as a positive control and E. faecalis strain SS-1273 is used as a negative control. QC is performed with each new lot number and shipment.

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6.5% NaCl Tolerance Test

I. Principle
Tolerance tests can be used in the differentiation of microorganisms. Some bacteria can grow in 6.5% NaCl and others are inhibited by these concentrations. Growth in broth containing 6.5% NaCl is determined in heart infusion broth base with the addition of 6% more NaCl. Heart infusion base contains 0.5% NaCl. To make the test easier to read we add 0.5% dextrose and brom cresol purple indicator.
II. Inoculum
A fresh inoculum grow in Todd Hewitt broth is preferred. Alternatively, a small loopful of growth from a blood agar plate may be used.
III. Reagents and Materials
6.5% NaCl broth 5 ml Todd Hewitt broth The 6.5% NaCl broth is prepared by the CDC Central Services Laboratory, formula No. 1707. This formulation is identical to the modified 6.5% NaCl broth described in: Facklam, R. 1973. Comparison of several laboratory media for presumptive identification of enterococci and group D streptococci. Appl. Microbiol. 26:138-145.
IV. Procedure
1. One or two colonies or one or two drops of an overnight broth culture is inoculated into the broth containing 6.5% NaCl.
2. The inoculated broth is incubated at 37ΕC in ambient air for up to a week or more depending upon the growth characteristics of the strain being tested. If 2 or 3 days were required for sufficient inoculum then the NaCl tolerance test should be incubated 10 to 14 days. In some cases (most enterococci) the test is positive after overnight incubation
V Reading and Interpretaion
When most strains grow the dextrose is fermented and the broth changes from purple to yellow color. However, the color change is not required for a positive test. If there is an obvious increase in turbidity, which indicates growth, without a change in color this is also read as a positive test.
VI Limitations
Other base media (brain heart infusion and trypticase soy) have been used for determining NaCl tolerance of the enterococci and viridans streptococci but these bases have not been tested with the other genera. Quality control
VII

Quality Control
Each new lot of media prepared by the CDC Central Services Laboratory is tested for positive (growth) and negative (no growth) reactions. Enterococcus faecalis strain SS-1273 and Streptococcus sanguinis strain SS-910 are used for positive and negative reactions respectively.

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Optochin Test

I. Principle
The purpose of the optochin test is to confirm the identification S. pneumoniae before serotyping and to aid in the differentiation of S. pneumoniae from viridans streptococci during surveillance studies.
II. Inoculum
Isolated alpha-hemolytic colonies suspected of being pneumococci
III. Reagents and Materials
1. optochin AP@ disks purchased from Becton Dickinson Microbiology Systems, Cockeysville, Md.
2. blood agar plates
IV. Procedure
1. Transferred an isolated colony and streak to a quarter of a blood agar plate
2. Place the optochin “P” disk in the upper third of the inoculum. Tap the disk to insure that it stays on the media after the plate is inverted.
3. The plate is incubated overnight at 35-37ΕC in a candle extinction jar or carbon dioxide incubator.
V Reading and Interpretaion
If a 6 mm disk is used, a zone of inhibition of at least 14 mm in diameter is considered positive for identification of pneumococci. A zone of inhibition between 6 and 14 mm in diameter is considered questionable for identification of pneumococci and a bile solubility test should be performed. Bile soluble strains with optochin zones of inhibition between 6 and 14 mm are considered pneumococci, those strains that are not bile soluble with the same zone sizes are not considered pneumococci.
VI Limitations
Cultures do not grow as well in normal atmospheres and larger zones of inhibition can cause misidentification.
VII

Quality Control
Each new lot and shipment of optochin AP@ disks is tested for positive and negative reactions. S. pneumoniae strain ATCC-49619 is inhibited by optochin (positive reaction) and S. mitis strain SS-429 is not inhibited by optochin (negative reaction). Results are recorded in the QC log book.

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Pigmentation Test

I. Principle
Some bacteria produce pigment. The purpose of the pigmentation test is to aid in the identification of E. casseliflavus, E. mundtii,E. pallens, E. gilvus and E. sulfureus. These enterococci produce a yellow pigment that can be detected on several different media.
II. Inoculum
The unknown Enterococcus strain is grown on trypticase-soy 5% sheep blood agar plate for 24 h in a normal atmosphere at 35ΕC.
III. Reagents and Materials
1. cotton swab
IV. Procedure
1. Use a cotton swab to pick up a 50 mm smear of bacteria.
2. Examine the swab and smear for a bright yellow color.
V Reading and Interpretaion
A pale to bright yellow color is interpreted as positive. A cream, white, or grey color is negative.
VI Limitations
The test should be repeated on the culture again after 48 h of incubation if there is any question about the results at 24 h.
VII

Quality Control
E. casseliflavus strain SS-1229 is used for a positive control and E. faecalis strain SS-1273 is used for a negative control for determining pigmentation.

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Pyridoxal Requirement Test (Vitamin B6)

I. Principle
Nutritionally variant streptococci (Abiotrophia and Granulicatella) are usually very fastidious and will grow only on supplemented media or enriched chocolate agar. These strains require pyridoxal for growth while non-NVS do not. A final concentration in broth of 0.001% of pyridoxal will support the growth of NVS. An alternative to performing the pyridoxal requirement test is the satellite test.
II. Inoculum
The cultures are typically received on chocolate agar slants.
III.

Reagents and Materials

1. 0.01% pyridoxal*
2. 2-TSA-sheep blood agar plate

* It is convenient to keep a 0.01% solution of pyridoxal in the laboratory. This solution is prepared in purified water and filter sterilized. A 10 ml aliquot is dispensed into sterile tubes. This 0.01% solution should be kept frozen at -20Ε C. The aliquot in use may be stored at 4° C.

IV.

Procedure

1. Add a drop of the 0.01% solution onto the surface of one of the TSA-sheep blood agar plates.
2. Inoculate both plates with the suspected NVS strain.
3. Incubate at 35°C in CO2 for 24 to 72 hours.

 
V Reading and Interpretaion
If growth is observed on the plate containing pyridoxal and not on the plate without pyridoxal the strain is a NVS.
VI Limitations
VII

Quality Control
There are no guidelines for quality control of the reagents or the media. Each lot of pyridoxal can be tested for supporting the growth of a known NVS strain.

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Pyrrolidonylarylamidase Test (PYR)

I. Principle
Some bacteria produce pyrrolidonyl arylamidase which hydrolyzes the substrate L- pyrrolidonyl -β-naphthylamide to form β-naphthylamine. A pink to red color forms when p-dimethylaminocinnam-aldehyde (PYR reagent) is added to β-naphthylamine.
II. Inoculum
Strains are grown on blood agar plates overnight at 35°C in CO2 . More than 1 day of incubation may be necessary for more fastidious genera such as the Gemellae, alloiococci, and helcococci. The strains to be tested are grown on a blood agar plate until sufficient growth is seen to heavily inoculate the disks.
III. Reagents and Materials
PYR disk (Remel)
PYR reagent
Loops
Deionized Sterile water
IV.

Procedure
The procedure that is used in the Streptococcus laboratory is modified from the package insert. The LAP test is usually done simultaneously.

1. Place the disks on blood agar plate in an area of little or no growth or on a slide. The moisture from the plate is usually sufficient to rehydrate the disk. If the disk is placed on a slide, then a tiny drop of sterile deionized water is added. (DO NOT OVERSATURATE THE DISK).
2. Using a loop or wooden stick, inoculate the disks heavily. Using two or more loop-fulls of culture is necessary for satisfactory results.
3. Leave the plates with the disks on the bench at room temperature for 10 minutes.
4. Add the detection reagent and read after 3 minutes.

 
V Reading and Interpretaion
The development of a red color within 3 minutes is positive. No change in color or a yellow color is negative. The color develops immediately. Discard the test after 10 minutes.
VI Limitations
False negative reactions may result if too little inoculum is used.
VII

Quality Control
Each lot and shipment of PYR disks are tested for positive and negative reactions. Enterococcus faecalis strain SS-498 is used for positive reaction and Streptococcus sanguinis strain SS-910 is used for a negative reaction.

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Pyruvate Utilization Test

I. Principle
Some bacteria possess the ability to utilize pyruvate which results in a change in pH. Bromthymol blue is added to the media as an indicator which results in a color change from blue-green to yellow.
II. Inoculum
A fresh inoculum grown in Todd Hewitt broth is preferred. Alternatively, a small loopful of growth from a blood agar plate may be used.
III. Reagents and Materials
Pyruvate broth (CDC Central Services Laboratory, formula # 1722)
IV. Procedure
1. Incoculate the pyruvate broth with one drop of an overnight Todd-Hewitt broth culture of the unknown strain.
2. Incubate the tube at 35ΕC for 7 days in ambient air. Fastidious strains may be incubated for 14 days.
V Reading and Interpretaion
A positive reaction is indicated by the development of a yellow color. If the broth remains green or greenish yellow, the test result is negative. A yellow color with only a hint of green is usually a positive reaction.
VI Limitations
VII

Quality Control
Quality control tests are determined for each new batch of prepared medium. E. faecalis strain SS-1273 and S. sanguinis strain SS-910 are used as positive and negative controls respectively. Results are recorded in the QC log book.

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Satellite Test (SAT)

I. Principle
Nutritionally variant streptococci (Abiotrophia and Granulicatella) are usually very fastidious and will grow only on supplemented media or enriched chocolate agar. The purpose of the SAT test is to determine if the unknown streptococcal strain is a nutritionally variant streptococci (NVS). These strains require pyridoxal for growth. Growth of Staphylococcus aureus on a TSA sheep blood agar plate releases these factors allowing NVS to grow. An alternative to performing the SAT test is to test for the requirement of vitamin B6 (pyridoxal).
II. Inoculum
The cultures are typically received on chocolate agar slants.
III. Reagents and Materials
1. Staphylococcus aureus ATCC-25923
2. TSA-sheep blood agar plate
IV. Procedure
1. Streak the submitted culture over the entire surface of a TSA-sheep blood agar plate with an inoculating loop.
2. A single streak of Staphylococcus aureus ATCC-25923 is then made across the middle of the agar plate.
3. Incubate the plate in a candle extinction jar or C02 incubator for 24 h or more.
V Reading and Interpretaion
If the strain is a NVS growth will appear only adjacent to the staphylococcus streak, about 2 to 5 mm wide. Streptococci that are not NVS will grow over the entire surface of the agar plate.
VI Limitations
VII

Quality Control
There are no quality control guidelines for testing the medium or the Staphylococcus strain for the satillitism test.

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Starch Hydrolysis Test

I. Principle
Some bacteria are able to hydrolyze starch on starch supplemented agar. When iodine is added to starch, it turns dark bluish-black. If starch has been hydrolyzed, then it is not available to react with the iodine and the area around the bacterial growth is clear. This test can be used to differentiate some bacteria.
II. Inoculum
Strains are grown on blood agar plates overnight at 35°C in CO2 . More than 1 day of incubation may be necessary for more fastidious genera such as the Gemellae, alloiococci, and helcococci. A drop of Todd Hewitt broth bacterial suspension may also be used to streak the plate.
III.

Reagents and Materials
1. Two percent starch agar (CDC Central Services Laboratory, formula #1710.)
2. Gram’s iodine

IV. Procedure
1. Inoculate a starch agar plate with a heavy single streak of a fresh culture or run a drop of broth culture across the plate.
2. Incubate the plate in CO2 at 35ΕC for 48 h. Some strains may require longer incubation for sufficient growth.
3. After incubation, flood the plate with Gram’s iodine.
V Reading and Interpretaion
A clear zone surrounding the growth is positive test that the strain hydrolyzed starch. A deep purple to black or bluish color of the agar indicates that starch has not been hydrolyzed and thus a negative test. For negative tests the deep color develops in the agar right up to the growth.
VI Limitations
Some fastidious strains show poor growth.
VII

Quality Control
Each new batch of starch agar is quality control tested. S. bovis strain SS-1224 is used for a positive control and E. avium strain SS-817 is used for a negative control.

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5% Sucrose Agar Slime Formation

I. Principle
The purpose of growing the unknown strains on agar containing 5% sucrose is to determine the capacity of the strains to form extracellular polysaccharide (levans or dextrans) on the agar.
II. Inoculum
Strains are grown on blood agar plates overnight at 35°C in CO2 . More than 1 day of incubation may be necessary for more fastidious genera such as the Gemellae, alloiococci, and helcococci. A drop of Todd Hewitt broth bacterial suspension may also be used to streak the plate.
III. Reagents and Materials
5% sucrose agar plates (CDC Central Services Laboratory, formula # 1714)
IV. Procedure
1. Inoculate the 5% sucrose agar with a fresh inoculum and streak for isolated colonies.
2. Incubate the agar plate in CO2 at 35ΕC for 48 h. Some fastidious strains may require longer incubation for growth.
3. Examine the plate for levans and dextrans. A loop is used to scrape the colonies for viscosity or adherence.
V Reading and Interpretaion
Some bacteria produce a levan as the extracellular polysaccharide. The colonies appear very slimy, mucoidal and runny or as large gum drops on the agar. Some bacteria may produce dextrans in which the colonies are dry and adherent to the plate. A negative reaction is the failure to see extracellular material on the 5% sucrose agar by visual inspection or adherence with a loop.
VI Limitations
VII

Quality Control
Quality control tests are performed on each batch of 5% sucrose agar. The following strains are use for QC: Leuconostoc mesenteroides strain SS-1238 (positive for slime production-levans), Streptococcus sanguinis SS910 (positive for dextrans-adherent) and Enterococcus faecalis strain SS-1273 (negative for levans and dextrans). Results are recorded in the QC log book.

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5% Sucrose Broth

I. Principle
The purpose of growing the unknown strains on agar containing 5% sucrose is to determine the capacity of the strains to form extracellular polysaccharide (levans or dextrans) in the broth.
II. Inoculum
Strains are grown on blood agar plates overnight at 35°C in CO2 . More than 1 day of incubation may be necessary for more fastidious genera such as the Gemellae, alloiococci, and helcococci. A drop of Todd Hewitt broth bacterial suspension may also be used to streak the plate.
III. Reagents and Materials
5% sucrose broth tubes (CDC Central Services Laboratory)
IV. Procedure
1. Inoculate the 5% sucrose broth with a fresh inoculum.
2. Incubate broth at 35ΕC for 48 h or longer. Some fastidious strains may require longer incubation for growth.
3. Examine the broth for viscosity or a gel button.
V Reading and Interpretaion
Some bacteria produce a levan as the extracellular polysaccharide. The broth appears very thick and slimey. Some bacteria may produce dextrans in which a gel button adhers to the bottom or sides of the tube. A negative reaction is the failure to see viscosity or adherence in the 5% sucrose broth by visual inspection. There is only an increase in turbidity.
VI Limitations
VII

Quality Control
Quality control tests are performed on each batch of 5% sucrose both. The following strains are use for QC: Leuconostoc mesenteroides strain SS-1238 (positive for slime production-levans), Streptococcus sanguinis SS910 (positive for dextrans-adherent gel button) and Enterococcus faecalis strain SS-1273 (negative for levans and dextrans). Results are recorded in the QC log book.

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Tellurite Tolerance Test

I. Principle
Tolerance to tellurite is determined on agar medium containing 0.04% potassium tellurite. Very few catalase-negative gram-positive cocci will grow on this medium. The primary purpose of the tellurite tolerance test is aid in the differentiation of E. faecalis and E. faecium and the other enterococci.
II. Inoculum
Strains are grown on blood agar plates overnight at 35°C in CO2 . More than 1 day of incubation may be necessary for more fastidious genera such as the Gemellae, alloiococci, and helcococci. A drop of Todd Hewitt broth bacterial suspension may also be used to streak the slant.
III. Reagents and Materials
1.Tellurite agar slants (0.04% potassium tellurite, CDC Central Services Laboratory, formula # 9358)
IV. Procedure
1. Inoculate the slant with one drop of a fresh (18-24 h) Todd-Hewitt broth culture or loopful of bacteria from a fresh plate.
2. Incubate at 35ΕC for 7 days in ambient air.
V Reading and Interpretaion
Tolerance (a positive result) is indicated whenever black colonies form on the surface. Typical and variant strains of E. faecalis usually form black colonies (positive tolerance) after 48 h of incubation. Some strains of E. faecium may form grey colonies (a negative reaction) but most stains fail to grow on tellurite medium. A slight blackening at the bottom of the slant is a negative result.
VI Limitations
VII

Quality Control
Quality control tests are performed on each new batch of medium supplied. E. faecalis strain SS-1273 and S. sanguinis strain SS-910 are used as positive and negative controls respectively. Results are recorded in the QC log book.

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Urea Hydrolysis

I. Principle
Urea provides a source of nitrogen for bacteria producing urease. The resulting change in pH causes the indicator, phenol red, to change from yellow to a red to pink-red color. The urease test is to particularly useful to aid in the identification of Streptococcus salivarius.
II. Inoculum
Strains are grown on blood agar plates overnight at 35°C in CO2 . More than 1 day of incubation may be necessary for more fastidious genera such as the gemellae, alloiococci, and helcococci. A drop of Todd Hewitt broth bacterial suspension may also be used to streak the slant.
III. Reagents and Materials
1. Christensen's urea agar (Remel)
IV. Procedure
1. Inoculate slant with a loopful or drop of a fresh culture.
2. Incubated at 35Ε C for up to 7 days in ambient air. Fastidious strains are incubated 14 day.
V Reading and Interpretaion
A positive reaction is recorded when a light or dark pink color develops in the agar slant. A yellow color or no change in the straw colored slant indicates a negative test.
VI Limitations
False positives may result due to protein hydrolysis but this has not been observed with the Streptococcus.
VII

Quality Control
Quality control is performed with each lot and shipment.S. salivarius, SS-908 and E. avium, SS-817 are used for positive and negative controls respectively.

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Vancomycin Test

I. Principle
The vancomycin test is performed in a fashion similar to the bacitracin sensitivity test. Resistance to vancomycin can be used to differentiate a few of the catalase-negative gram-positve genera.
II. Inoculum
Strains are grown on blood agar plates overnight at 35°C in CO2 . More than 1 day of incubation may be necessary for more fastidious genera such as the Gemellae, alloiococci, and helcococci.
III. Reagents and Materials
1. 30µg vancomycin disk (Becton Dickinson Microbiology Systems, Cockysville Md. Product No. 31353) The disks are stored according to the manufacturer's instructions.
2. trypticase soy sheep blood agar plates
IV. Procedure
1. Transfer several colonies of the strain in question to one-half of a blood agar plate and steak heavily.
2. Place the vancomycin susceptibility testing disk (30 µg) in the heavy part of the streak.
3. Incubated the plate in a CO2 enhanced atmosphere at 37ΕC overnight. Some strains (alloiococci, gemellae, helcococci) may require 48 h or more to show sufficient growth to interpret the test.
V Reading and Interpretaion
Any zone of growth inhibition is considered positive (sensitive). The test is interpreted as resistant (negative) only if there is growth right up to the edge of the disk. This is not a susceptibility test, it is a sensitivity test for identification.
VI Limitations
Sufficient inoculum is required for an accurate test.
VII

Quality Control
Each new lot and shipment of vancomycin disks is tested for positive (sensitive) and negative (resistant) reactions. Streptococcus bovis SS1224 is used as a positive (sensitive) reaction and Leuconostoc mesenteroides strain SS-1238 (ATCC-8293) is used for the negative (resistant) reaction. Results are recorded in the QC log book.

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Voges-Proskauer Test (VP)

I. Principle
The Colbentz modification of the Voges-Proskauer (VP) test can be used to determine the production of acetylmethyl carbinol. Test reactions are used for differentiation of bacteria. The purpose of the VP test is to aid in the identification of Streptococcus anginosus strains of ß-hemolytic streptococci (Table 2). The VP test can also be used to aid in the differentiation of the viridans streptococci into Species/groups (Table 6).
II. Inoculum
A fresh inoculum grown in Todd Hewitt broth is preferred. Alternatively, a small loopful of growth from a blood agar plate may be used.
III. Reagents and Materials
1. Voges-Proskauer (VP) test broth (Remel)
2. 40% KOH (bioMerieux)
3. alpha-naphthol (bioMerieux)
IV. Procedure
1. Inoculate VP broth tube with one drop or loopful of fresh inoculum.
2. Incubate 24-48 hrs at 35°C in ambient air or until turbid growth is observed.
3. Transfer 0.5 ml to a 13X100 glass test tube.
4. Add twelve drops alpha naphthol and 4 drops 40% KOH.
5. Carefully vortex or shake tubes and observed for 30 minutes. The tube must be vigorously shaken several times during the 30 minute period.
V Reading and Interpretaion
A positive reaction, a red color, is usually seen within 30 minutes. For streptococcal identification weak reactions (pink or rust colors) are interpreted as positive.
VI Limitations
VII Quality Control
Each lot and shipment of media and reagents are tests. Enterococcus faecalis SS1273 is used as the positive control and Streptococcus sanguinis SS910 is used as the negative control. Results are recorded in the QC log book.

 

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Page last modified: September 6, 2006
Content source: National Center for Immunization and Respiratory Diseases
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