Method for differentiating microorganisms in a sample

BACKGROUND OF THE INVENTION
Differentiation and identification of microorganisms such as bacteria, fungi or yeasts in a sample suspected of containing pathogenic organisms is important. The non-pathogenic organisms must be distinguished from the pathogens. Most frequently, samples contain mixed bacteria, mixed fungi or mixed yeasts, whether a clinically obtained sample, or an environmental sample, e.g. from a water supply, or a food based sample, including poultry and dairy products.
While numerous methods have evolved to help distinguish and identify microorganisms, most involve appropriate selection of culture media and several additional steps including incubating, preliminary identification by morphology, followed by further culturing and incubation of selected microorganisms. In some instances, complete identification of the microorganisms in a sample can take several days, as the cultures are typically incubated for 18-24 hours before significant growth is detected.
By way of example, by formulating the culture media to permit the growth of only bacteria with certain characteristics, the growth of some bacteria (which are not of interest) can be suppressed. However, regardless of how culture media are adjusted, by altering ingredients or the concentrations of ingredients, selection of the culture media still may not typically result in clear identification and differentiation of all the bacterial species present in the sample. Microscopic examination of morphology and further testing are commonly required, or use of multiple, differing culture media.
Use of color to differentiate various microorganisms was attempted against this backdrop.
While use of color to differentiate and identify microorganisms has been known for many years, until recently, these methods suffered from many drawbacks, including deterioration of the color change, diffusion or evaporation of the color formed, generation of color which cannot distinguish between bacteria or which is difficult to read against the color of the media, or the necessity for several steps, including UV radiation to develop the color.
Relatively recently, so-called chromogenic media have been utilized to differentiate microorganisms.
The primary method employed by chromogenic media to detect and differentiate microorganisms is the reaction of glycosidase substrates, as shown below. This basic chemical reaction to form color is exemplified by X-Gal (5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside), a reagent commonly used in molecular biology to monitor lacZ gene exprssion. This colorless substrate undergoes a reaction with galactosidase to produce an insoluble blue indigo product: ##STR1##
The oxidation step occurs rapidly to produce the indigo compound from the unstable 3-hydroxy indole. This oxidation may be air mediated and may also involve cellular metabolic processes in some organisms. One advantage of the indigo compounds as microbial indicators is their localization within the microorganisms, which helps to prevent deterioration and diffusion of the color change. The indigo compounds are also relatively nontoxic to growing microorganisms. Another advantage is that several colors are available by changing substituents on the indolyl ring. This allows differentiation of microorganisms with different glycosidase activities. A list of such colored substrate products is given below. ##STR2##
Products of known indolyl glycoside substrates:
______________________________________R.sub.1 R.sub.2 R.sub.3 R.sub.4 Color______________________________________H H H H blueCl Br H H blueH Br Cl H magentaH H Cl H salmonH I H H purpleH Br H H blue______________________________________
In addition to glycosidase substrates, dyes such as pH indicators are occasionally used in chromogenic media. Chromogenic indolyl substrates for esterases, phosphatases, and other enzymes are also used in some applications. These work similarly to the glycosidase substrates. An example, "Mag-Phos" is shown below. ##STR3##
Recently, many examples of testing methods utilizing this basic chemistry have been reported, some of which are described below.
Rapid identification of Salmonella is an important public health issue. Culture media for this purpose comprising a chromogenic compound linked to a C.sub.7 -C.sub.10 fatty acid, and an appropriate detergent which promotes liberation of the chromogenic compounds is proposed in WO94/09152. The chromogenic compound is preferably 5-bromo-4-chloro-3-indolyl caprylate, and the detergent is selected from fused polycyclic detergents. .beta.-glucosides and/or .beta.-galactosides are advantageously added.
Chromogenic compounds derived from indolylglucuronic acid as a substrate for the GUS enzyme are described in WO 94/08043.
Combining a chromogenic .beta.-galactosidase and a .beta.-galactosidase and a .beta.-glucoronidase in a test medium to distinguish between Escherichia coli and general coliforms in a single test with a single sample is disclosed in U.S. Pat. No 5,210,022.
A test medium useful for identifying bacteria found in urine samples containing a chromogenic .beta.-glucoronidase substrate capable of forming a first color when reacted with .beta.-glucoronidase, a chromogenic arylsulfatase substrate capable of forming a second color when reacted with arylsulfatase, and a nutrient base is described in U.S. Pat. No. 5,464,755. Proteinaceous opaque compounds, such as milk-derived compounds can be included in the test medium. A medium for isolating Salmonella colonies without ambiguity by use of specific coloration is disclosed in U.S. Pat. No. 5,194,374. This medium contains peptones, a polyol metabolizable by Salmonella and a pH indicator sensitive to acidification. The polyol is adsorbed on a pulverulent material. The medium may also contain deoxycholate and a chromogenic .beta.-galactosidase substrate.
A method for revealing the presence or absence of a particular microorganism strain, with at least one strain-specific enzyme substrate chromogen and at least one compound selected from a high concentration carbohydrate, are added to the culturing medium as disclosed in WO95/04156. Once the chromogen has been hydrolyzed, a color differing from the basic color of the chromophore results.
A method of identifying E. coli using a growth medium for E. coli with 8-hydroquinoline-.beta.-D-glucuronide as an activator such as X-glucuronide is disclosed in EP 0025467. This method shows the E. coli as darkly pigmented blobs.
Determining the presence of E. coli in a liquid sample passed through a suitable membrane filter by contacting the filter with a chromogenic reagent, indoxyl-.beta.-D-Glucuronide, in an E. coli nutrient medium followed by incubation is described in U.S. Pat. No. 4,923,804. The E. coli appears as an indigo blue color.
A method for identifying Enterobacteriaceae in a single culture medium is disclosed in U.S. Pat. No. 3,870,601. The media comprises a mixture of chromogenic .beta.-galactoside substrates with a decarboxylase substrate, a deaminase substrate, a urease subtrate, a hydrogen disulfide detection system, or a carbohydrate fermentation system. ONPG (o-nitrophenyl-.beta.-galactopyranoside) is disclosed as suitable.
Use of two different chromogens, and biological material capable of fermenting a sugar in a test medium adjusted to a pH conducive for color change of a pH indicator upon acidification upon fermentation of the sugar, is used to distinguish bacteria in WO 97/36001.
Direct detection of Salmonella (except S. arizonae) is possible by combining glucuronic acid and a pH indicator in the culture medium, together with a chromogenic or fluorogenic compound capable of being hydrolyzed by .beta.-galactosidase, as described in U.S. Pat. No. 5,434,056.
WO96/40861, like other art, is concerned with the identification of pathogens such as the possibly fatal E.coli 0157:H7 and Salmonella. A medium, liquid or solid, containing propionic acid, one or more chromogenic substrates, such as galactosidase substrates and glucuronidase substrates, and a nutrient base can identify these bacteria, according to this document.
Differentiation of pathogenic monocytogenes species of Listeria from the non-pathogens by use of a glycine amino peptidase substrate is disclosed in U.S. Pat. No. 5,330,889. Identification and differentiation of different species of yeasts can be accomplished using CHROMagar Candida plates available from CHROMagar Company, Paris, France. Yeasts from clinical samples grown on these plates are identified by variant colors and morphology. See e.g. A. P. Koehler, et al. J. Clin. Microbiol., 37, pp. 422-26 (1999). The CHROMagar medium is composed of 10 g peptone, 20 g glucose, 15 g agar, 0.5 g chloramphenicol, per liter, and a "chromogenic mixture," whose components are maintained in secrecy by the manufacturer. (E. T. S. Houang, et al., J. Clin. Path., 50, pp. 563-565 (1997).) The yeast colonies appear in colors such as pink, blue, apple green and rose on a clear background.
CHROMagar Salmonella (CAS) is used to identify Salmonella spp. as mauve colonies after 18 hours of incubation, while other members of the Enterobacteriaceae grow as blue or uncolored colonies. (O. Gaillot, et al., J. Clin. Microbiol., 37, pp. 762-65 (1999).)
While there are obviously many methods known and described for differentiating microorganisms, those currently known involve the use of culture media which are clear, thus providing good contrast and ready visibility when a color change takes place. Use of opaque media have also been used with chromogens, such as resulting from the addition of proteinaceous materials such as milk. Here again, however, the color change of susceptible bacteria is easy to detect.
Not all clinically important bacteria, yeasts or fungi can be cultured on the chromogenic media described above. For example, the so-called fastidious bacteria have specific growth requirements, and will not grow, (or grow in a meaningful way) on routine media. Examples of clinically important fastidious bacteria include Neisseria and Haemophilus. Many of these bacteria are found in respiratory, cerebral-spinal and genital fluids and secretions.
For these fastidious bacteria, incubation and growth is normally performed on chocolate agar, a culture medium containing hemoglobin. (See, Martin et al., Publ. Health Rep., 82:361 (1967). As its name implies, chocolate agar looks like chocolate and is brown in color. Therefore, whether chromogenic media will provide any meaningful contrast to permit differentiation and identification of bacteria is unknown.
Trypticase Soy Agar (TSA) with sheep blood is a culture media which is commonly used for the cultivation and isolation of fastidious microorganisms when distinct hemolytic reactions are important (e.g., Streptococcus pneumomiae from respiratory specimens). This culture media supports growth of many kinds of bacteria, which can only be distinguished by their morphology, which may be extremely difficult. Due to the presence of the blood, this media has a bright red color, and therefore it is unknown whether a color change would be discernible on this media.
The TSA with sheep blood media differentiates microorganisms on the basis of hemolysis, which results in the clearing (or lysis) of red blood cells due to the production of hemolysins by the microorganisms.
Due to their dark color it was unknown whether chromogenic reactions would be discernable on TSA and blood or chocolate agar plates. Moreover, it was unknown whether the presence of chromogens in the media would interfere with the hemolytic reaction on TSA with sheep blood media.
It is an object of this invention to prepare a chromogenic indicator medium containing blood or hemin for growing and identifying microorganisms.
It is another object of the present invention to prepare a TSA and sheep blood culture medium containing chromogenic substrates.
It is also an object of the present invention to differentiate microorganisms based on a change in color of the microorganisms on a TSA and sheep blood culture medium.
Another object of the present invention is to prepare a chocolate agar culture medium containing chromogenic substrates.
Yet another object of this invention is to differentiate bacteria based on a change in color of the bacteria on chocolate agar media.
SUMMARY OF THE INVENTION
The present invention relates to a method for differentiating microorganisms in a sample utilizing a chromogenic indicator medium containing blood or hemin for growing and identifying microorganisms.
The present invention also encompasses a method for utilizing a chromogenic indicator media for color differentiating microorganisms, including bacteria, yeasts and fungi, in a culture medium containing blood or hemin such as Trypticase Soy Agar with 5% Sheep Blood (TSASB), or Chocolate agar. Other dye or color producing compounds, such as crystal violet, could be used in the media as well. The resulting chromogenic reactions are visible and easily readable on these non-clear, i.e., colored growth media. Surprisingly, the presence of the chromogens do not adversely affect the other differential properties of the growth media, such as hemolytic reactions, colony size or shape or other characteristics. This invention can be used for clinical and industrial (e.g. food, water, environmental or pharmaceutical) specimens, and includes a method for differentiating bacteria on TSASB and Chocolate agar growth media with chromogens. The invention also includes methods for preparing chromogenic indicator media containing blood or hemin.
DETAILED DESCRIPTION OF THE INVENTION
The present invention uses chromogens in conjunction with known growth media TSASB and chocolate agar.
TSASB is commercially available. For example, pre-made plates containing this growth media can be obtained from Becton Dickinson Biosciences, Cockeysville, Md., under the name TSA II.TM.. Such plates are normally stable, under refrigeration, for 14-16 weeks.
Alternatively, TSASB can be made in according to known formulas and procedures. See, e.g. Dilworth, et al., J. Clin. Microbiol., 2:453 (1975).
Chocolate agar growth media are also commercially available, pre-plated and in tubed slants, from manufacturers such as Becton Dickinson Biosciences, Cockeysville, Md., under the name Chocolate Agar II.
Chocolate agar can also be made according to known formulas and procedures. See, e.g. Martin et al., Publ. Health Rep., 82:361 (1967).
As discussed in the Background herein, suitable chromogenic substrates are compounds which will be acted upon by enzymes found in the target bacteria to result in an insoluble colored product which is contained within the bacteria. Many such chromogenic substrates are known and each is targeted to a specific enzyme.
Examples of suitable chromogenic substrates are provided below. This listing is not meant to be all-inclusive. Other such compounds are known, and are available from companies such as Sigma (St. Louis, Ill.), INALCO (Milan, ITALY), BIOSYNTH (AG Staad, SWITZERLAND), BIOSYNTH INTERNATIONAL (Naperville, Ill.) and GLYCOSYNTH (Warrington, Cheshire, England).
______________________________________Abbreviation Chemical Name______________________________________X-Acglmn 5-Br-4-Cl-3-indolyl-N- Acetyl-.beta.-D-galactosaminideX-Cap 5-Br-4-Cl-3-indolyl- caprylateBG-Gal 4-Cl-3-indolyl-.beta.-D- galactopyranosideSal-Gal 6-Cl-3-indolyl-.beta.-D- galactopyranosideX-Gal 5-Br-4-Cl-3-indolyl-.beta.-D- galactopyranosideX-Glucoside 5-Br-4-Cl-3-indolyl-.beta.-D- glucopyranosideSal-Glucuro 6-Cl-3-indolyl-.beta.-D- glucuronide (CHA salt)X-Gluc 5-Br-4-Cl-3-indolyl-.beta.-D- glucuronide (CHA salt)X-Glucuro (c) 5-Br-4-Cl-3-indolyl-.beta.-D- glucuronide (CHA salt)X-Glucuro (s) 5-Br-4-Cl-3-indolyl-.beta.-D- glucuronide (sodium salt)IPTG Isopropyl-.beta.-D- thiogalactopyranosideMag-Phos 5-Br-6-Cl-3-indolyl- phosphate, p-touidine saltX-Sulfate 5-Br-4-Cl-3-indolyl-sulfate (Potassium salt)X-.alpha.-Glucoside 5-Br-4-Cl-3-indolyl-.alpha.-D- glucopyranosideX-Cello 5-Br-4-Cl-3-indolyl-.beta.-D- cellobiosideX-Acetate 5-Br-4-Cl-3-indolyl-3-______________________________________
______________________________________Abbreviation Chemical Name______________________________________ acetateX-Glucosamine 5-Br-4-Cl-3-indolyl-.beta.-D- glucosaminideX-Butyrate 5-Br-4-Cl-3-indolyl- butyrateX-Palminate 5-Br-4-Cl-3-indolyl- palminateX-Phosphate 5-Br-4-Cl-3-indolyl- phosphateX-Fucoside 5-Br-4-Cl-3-indolyl-.beta.-D- fucosideX-Xylo 5-Br-4-Cl-3-indolyl-.beta.-D- xylopyranoside______________________________________
Chromogenic substrates, such as those noted above, can be added to the growth media in at least two ways.
A small amount of the chromogenic substrate, from 0.05 to 0.2 g, preferably 0.05 to 0.1 g (e.g. 0.08 g) can be added to a small amount of DMSO (e.g. 1 ml.). Then a small aliquot of this solution (e.g. about 50 microliters) can be added to the surface of a pre-plated medium, either TSASB or chocolate agar, then distributed using a spreader. The plate is then allowed to dry 3-4 hours in a hood. It will be noted that in some instances a powdery insoluble precipitate forms during spreading.
Another method of incorporating the chromogenic substrates into the growth media is to freshly prepare the media in accordance with known procedures. All chromogens can be added aseptically to the base post autoclaving at a preferred concentration of 0.1 g/l (a suitable range is 0.05 to 0.20 g/l ). Chromogens are either pre-dissolved in DMSO or added as powder, if water soluble. Chromogens can also be added prior to autoclaving.
Other methods for incorporating the chromogenic substrates will be apparent to those of ordinary skill in the art.
The fungi which may be used in the present invention are those known to one of ordinary skill in the art and include Aspergillus spp., Trichosporn spp. and Geotrichum spp. Likewise, yeasts which may be evaluated in the present invention are known to those of ordinary skill and include Candida spp. and Cryptoccus spp.
The bacteria to be grown and differentiated using the present invention are those which are known to be suitable for growth on blood or hemin containing media. As noted previously, fastidious bacteria are intended for the Chocolate agar medium. Examples include Haemophilus spp. and Neisseriae spp.
Sources of such bacteria include clinical samples, such as sputum, urine and blood and industrial sources, such as food, water, environmental or pharmaceutical samples.
TSASB suitable bacteria are also well known. Examples are Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae.
Samples of suitable bacteria for TSASB can be obtained from many sources, including clinical samples and industrial samples.
Of course, as is well known, specific, identified bacteria can be obtained from sources such as ATCC, the American Type Culture Collection in Manassas, Va.
Specific examples of the use of various chromogenic substrates on both TSASB and chocolate agar media are provided below. The specific experiments set forth below demonstrate that various bacteria respond differently to the presence of chromogenic substrates.
Some bacteria will develop a color, such as dark blue, green or pink, that will differentiate them, perhaps in combination with a characteristic morphology, from other bacteria in a sample. In some cases, a particular bacteria may remain colorless, while others of the same species do develop color, thus providing another basis to differentiate the bacteria. Yet another possibility is that a particular bacterium will develop a color different from those of other bacteria in a sample. This provides yet another means for the bacteria to be differentiated from other bacteria in a sample.

EXAMPLE 1
In this series of experiments, 0.08 g of each chromogen indicated below was added to 1.0 ml DMSO and thoroughly mixed. All the chromogens appeared to be soluble, providing a clear and/or colorless solution, except for Mag-Phos, which appeared clear and light orange in color.
______________________________________Abbreviation Chemical Name______________________________________X-Acglmn 5-Br-4-Cl-3-indolyl-N- Acetyl-.beta.-D-galactosaminideX-Cap 5-Br-4-Cl-3-indolyl- caprylateBG-Gal 4-Cl-3-indolyl-.beta.-D- galactopyranosideSal-Gal 6-Cl-3-indolyl-.beta.-D- galactopyranosideX-Gal 5-Br-4-Cl-3-indolyl-.beta.-D- galactopyranosideX-Glucoside 5-Br-4-Cl-3-indolyl-.beta.-D- glucopyranosideSal-Glucuro 6-Cl-3-indolyl-.beta.-D- glucuronide (CHA salt)X-Glucuro (c) 5-Br-4-Cl-3-indolyl-.beta.-D- glucuronide (CHA salt)X-Glucuro (s) 5-Br-4-Cl-3-indolyl-.beta.-D- glucuronide (sodium salt)IPTG Isopropyl-.beta.-D- thiogalactopyranosideMag-Phos 5-Br-6-Cl-3-indolyl- phosphate, p-touidine saltX-Sulfate 5-Br-4-Cl-3-indolyl-sulfate (Potassium salt)______________________________________
50 .mu.l of each chromogen solution was added to the surface of each of the following pre-plated media (one chromogen solution per plate):
TSAII 5% Sheep Blood;
Chocolate II; and
TSA plain (no blood).
All pre-plated media were obtained from Becton-Dickinson Microbiology Systems, MD.
The solution was dispersed using a spreader and allowed to dry 3-4 hours in a hood. In some instances a powdery, insoluble precipitate formed during spreading, except for X-Glucuro(s) which remained clear. After 1 week, however, some precipitate was evident only on the X-Glucoside and X-Acglmn plates.
The bacterial test strains were adjusted to a 0.5 McFarland equivalent and diluted 1:10 in sterile saline. The plates were then inoculated using the standard streak plate method.
The following bacteria were evaluated: Escherichia coli 25922; Staphylococcus aureus 25923; S. epidermidis 12228; Streptococcus pneumoniae 6303; Group B Strep. 12386; Haemophilus influenzae 35540, 10211; Branhamella catarrhalis 25238; Neisseria meningitidis 13090; N. sicca 29193; N. gonorrhoeae 35201; and Gardnerella vaginalis 14019.
All plates were incubated at 35.degree. C. for 20-24 hours. The TSAII with Sheep blood and TSA II (plain) were incubated in air, while the chocolate agar was incubated in CO.sub.2 (5%).
After incubation, the plates were evaluated for growth by quadrant readable colonies.
The fastidious strains of bacteria were only tested on Chocolate II Agar. Results of these tests are presented below in Tables I and II.
TABLE I__________________________________________________________________________Evaluation of Microorganism Colony Color Formation Following Growth onTSA IIWith 5% Sheep Blood, TSA plain and Chocolate II Agar Supplemented WithVarious Chromogenic Substrates__________________________________________________________________________ TSA Plain TSA II 5SB Choc IITest Strain Substrate Growth Color Growth Color Growth Color__________________________________________________________________________S. epi X-Acglmn 4+ WT 4+ WT 4+ WT12228 X-Cap NG NA 1+ WT NG NA X-Gal 1+ WT 4+ WT 4+ WT X-Glucoside 4+ WT 4+ WT 4+ WT X-Glucuro 4+ WT 4+ WT 4+ WT Mag-phos 1+ purple 1+ purple 1+ purpleE. coli X-Acglmn 4+ NC 4+ NC 4+ NC25922 X-Cap 4+ NC 4+ NC 4+ NC X-Gal 4+ blue 4+ blue 4+ blue X-Glucoside 4+ NC 4 NC 4+ NC X-Glucuro 4+ blue 4+ blue 4+ blue Mag-Phos 4+ purple 4+ purple 4+ purpleS. aureus X-Acglmn 4+ NC 4+ NC 4+ NC25923 X-Cap 1+ turquoise 4+ turquoise 4+ turquoise (mixed) mixed X-Gal 4+ NC 4+ NC 4+ NC X-Glucoside 4+ NC 4+ NC 4+ NC X-Glucuro 4+ NC 4+ NC 4+ NC Mag-Phos 1+ purple 3+ purple 2-3+ purpleS. pneu: X-Acglmn 1+= NC 4+= blue 4+= NC6303 X-Cap NG NA < NC NG NC X-Gal = NC = light blue = NC X-Glucoside < NC = NC = NC X-Glucuro = NC = NC = NC Mag-Phos NG NA < NC < NCB. catt X-Acglmn = NC = NC = NC25258 X-Cap NG NA < NC < NC X-Gal NG NA = NC = NC X-Glucoside < NC = NC = NC X-Glucuro >> NC = NC = NC Mag-phos NG NA << NC NG NCGroup B X-Acglmn = NC = NC = NCStrep 12386 X-Cap NG NA < NC << NC X-Gal < NC < NC = NC X-Glucoside = blue = blue = blue X-Glucuro = blue = blue (a) = blue Mag-phos < purple < purple (a) = purple__________________________________________________________________________ Substrates on Chocolate II AgarTest Strain X-Acglmn X-Cap X-Gal X-Glucoside X-Glucuro Mag-Phos__________________________________________________________________________H. influ. = NC = NC = NC = NC = NC = purple35540N. gono. = NC NG < NC < NC NG NG35201G. vag. 14019 = NC NG = NC < NC NG NGH. influ. = NC NG = NC = NG = NC = purple10211N. sicca. = NC blue = NC = NC = NC = purple29193N. mening. < NC NG = NC = NC = NC = NC13090__________________________________________________________________________ Key: NC = no Color a = not as hemolytic NG = no growth WT = white NA = not applicable = growth equivalent to control sample < colony size/quadrant less
TABLE II______________________________________Summary of Table I Results Substrates Producing Substrates ProducingTest Strains Color Growth Inhibition______________________________________E. coli 25922 X-Gal, X-Glucuro, None Mag-phosS. aureus 25923 X-Cap, Mag-phos NoneS. epidermidis 12228 Mag-phos X-CapS. pneumoniae 6303 X-Acglmn (TSA II X-Cap, Mag-phos only)Group B Strep. 12386 X-Glucoside, X- Slight inhib. W/ X- Glucuro, Mag-phos Cap, X-Gal, Mag-phosH. influenzae 35540, Mag-phos None10211B. catarrhalis 25238 None X-Cap, Mag-PhosN. meningitidis None Slight inhib w/ X-13090 AcglmnN. sicca 29193 X-cap, Mag-phos NoneN. gonorrhoeae 35201 None X-Cap, X-Glucuro, Mag-phosG. vaginalis 14014 None X-Cap, X-Gal, X- Glucuro, Mag-phos______________________________________
It was surprising to discover that chromogenic substrates dissolved in the organic solvent dimethylsulfoxide (DMSO), when applied to the surface of or incorporated within a highly colored medium (i.e., a medium containing blood or hemin) could support the growth of microorganisms and result in color differentiation of the microorganisms.
The Haemophilus influenzae, both strains, appear dark purple on chocolate agar with Mag-phos. This is clearly differentiated from other sputum normal flora which appeared non-colored.
Group B streptococcus appears blue with beta hemolysis on TSASB with X-Glucuro.
Neisseria sicca appears purple on Chocolate agar with Mag-phos. N. meningitidis appears colorless.
In conducting these experiments, it was found that X-Cap and Mag-phos were somewhat insoluble, forming a white precipitate, when placed on the plated media. These two compounds also proved to have the most inhibitory effect. It is possible the concentration used for these compounds was too high.
EXAMPLE 2
Using the procedure of Example 1, the bacteria tabulated below were evaluated on TSA II 5% Sheep Blood and Chocolate II agar. In each instance, the chromogens were added to the surface of the prepared-plated media at 0.004 g/plate in 50 .mu.l DMSO. The following bacteria were evaluated: Branhamella catarrhalis; Clostridium jejuni; Clostridium perfringens; Escheria coli; Enterococcus faecalis, Peptostreptococcus spp.; Streptococcus agalactiae; Staphylococcus aureus; Staphylococcus epidermidis; Streptococcus mitis; Streptococcus pneumoniae; Streptococcus pyogenes; and Streptococcus groups C, F and G.
The results of this experiment, designed to evaluate the effect of growth conditions (incubation) on colony color formation, are tabulated below:
TABLE III__________________________________________________________________________TSA II 5% Sheep Blood (chromogens added to surface at 0.004 g/plate in 50.mu.l DMSO) Reference IncubationStrain No. Conditions X-Acglm X-Cap X-Gal X-Glucoside X-Glucuro Mag-Phos__________________________________________________________________________B. catarrhalis 25258 24 h, CO.sub.2 NC NC NC NC NC NGC. jejuni 33291 5 d, MA NC NG NC NC NC NGC. jejuni 33292 5 d, MA NC NG NC NC NC NGC. perfringens 13124 5 d, ANA blue (l) NG blue (m) NC NC NGE. coli 25922 24 h, air NC NC blue (d) NC blue (d) purple (m)E. coli 25922 5 d, ANA NC NC blue (d) NC blue (m) purple (m)E. coli 25922 5 d, MA NC NC blue (d) NC blue (m) purple (m)E. faecalis 29212 24 h, CO.sub.2 NT NT NT NT NC NTPeptostrep. spp 27337 5 d, ANA NC NG NC NC NC NGS. agalactiae 4768 24 h, CO.sub.2 NT NT NT NT blue (l) NTS. agalactiae 6638 24 b, CO.sub.2 NT NT NT NT blue (m) NTS. agalactiae 12386 24 h, CO.sub.2 NC NC NC blue (d) blue (d) purple (m)S. agalactiae 12386 24 h, CO.sub.2 NT NT NT NT blue (d) NTS. agalactiae 13813 24 h, CO.sub.2 NT NT NT NT blue (d) NTS. agalactiae BD11586 24 h, CO.sub.2 NT NT NT NT NC NTS. agalactiae BD747 24 h, CO.sub.2 NT NT NT NT grey blue NTS. aureus 25923 24 h, air NC blue (l) NC NC NC purple (m)S. epidermidis 12228 24 h, air NC NC NC NC NC purple (m)S. mitis 6249 24 h, CO.sub.2 blue (vl) NT NT NT NT NTS. pneumoniae 6303 24 h, CO.sub.2 blue (l) NC blue (l) NC NC NCS. pyogenes 19615 24 h, CO.sub.2 NT NT NT NT NC NTStrep Grp C 12388 24 h, CO.sub.2 NT NT NT NT blue (d) NTStrep Grp F 12392 24 h, CO.sub.2 NT NT NT NT NC NTStrep Grp F BD77 24 h, CO.sub.2 NT NT NT NT NC NTStrep Grp G 12394 24 h, CO.sub.2 NT NT NT NT grey blue NT (l)Strep Grp G 27961 24 h, CO.sub.2 NT NT NT NT blue (d) NT__________________________________________________________________________ Abbrev: NC = No Color 24 h = 24 hours NT = Not Tested 5 d = 5 days NG = no growth CO.sub.2 = 5% CO.sub.2 vl = very light MA = microaerophilic; 5-12% CO.sub.2 and 5-15% O.sub.2 l = light ANA = anaerobic m = medium d = dark
TABLE IV__________________________________________________________________________Chocolate II Agar (chromogens added to surface at 0.004 g/plate in 50.mu. DMSO Reference IncubationStrain No. Conditions X-Acglm X-Cap X-Gal X-Glucoside X-Glucuro Mag-Phos__________________________________________________________________________B. catarrhalis 25258 24 h, CO.sub.2 NC NC NC NC NC NGE. coli 25922 24 h, air NC NC blue (d) NC blue (d) purple (m)E. faecalis 29212 24 h, CO.sub.2 NT NT NT NT NC NTG. vaginalis 14019 24 h, CO.sub.2 NC NG NG NC NG NGH. influenzae 10211 24 h, CO.sub.2 NC NC NC NC NC purple (m)H. influenzae 35540 24 h, CO.sub.2 NC NC NC NC NC purple (m)N. 35201 24 h, CO.sub.2 NC NG NC NC NG NGgonnorhoeaeN. meningitidis 13090 24 h, CO.sub.2 NC NG NC NC NC NC (col dark)N. sicca 29193 24 h, CO.sub.2 NC blue (m) NC NC NC purple (m)S. agalactiae 4768 24 h, CO.sub.2 NT NT NT NT blue (l) NTS. agalactiae 6638 24 h, CO.sub.2 NT NT NT NT blue (m) NTS. agalactiae 12386 24 h, CO.sub.2 NC NC NC blue (d) blue (d) purple (m)S. agalactiae 12386 24 h, CO.sub.2 NT NT NT NT blue (d) NTS. agalactiae 13813 24 h, CO.sub.2 NT NT NT NT blue (d) NTS. agalactiae BD11586 24 h, CO.sub.2 NT NT NT NT blue (vl) NTS. agalactiae BD747 24 h, CO.sub.2 NT NT NT NT blue (l) NTS. aureus 25923 24 h, air NC blue (l) NC NC NC purple (m)S. epidermidis 12228 24 h, air NC NG NC NC NC purple (m)S. mitis 6249 24 h, CO.sub.2 NC NT NT NT NT NTS. pneumoniae 6303 24 h, CO.sub.2 NC NG NC NC NC NCS. pyogenes 19615 24 h, CO.sub.2 NT NT NT NT NC NTStrep Grp C 12388 24 h, CO.sub.2 NT NT NT NT blue (d) NTStrep Grp F 12392 24 h, CO.sub.2 NT NT NT NT NC NTStrep Grp F BD77 24 h, CO.sub.2 NT NT NT NT NC NTStrep Grp G 12394 24 h, CO.sub.2 NT NT NT NT blue (d) NTStrep Grp G 27961 24 h, CO.sub.2 NT NT NT NT blue (d) NT__________________________________________________________________________ Abbrev: NC = No Color CO.sub.2 = 5% CO.sub.2 NT = Not Tested NG = no growth vl = very light l = light m = medium d = dark
As will be observed from a review of the data tabulated above, X-Acglmn and X-Gal may be useful to differentiate C. perfringens from other Clostridia spp. grown under microaerphilic or anaerobic conditions. Also, X-Gal, X-Glucurono and Mag-Phos may be used to differentiate E. coli grown under microaerophilic or anerobic conditions, in addition to ambient air conditions.
EXAMPLE 3
In this series of experiments, the chromogenic substrates were added directly into the culture medium as it was being prepared and prior to distribution to plates.
Laboratory prepared media consisting of TSA II base and 5% sheep blood were prepared in accordance with the procedures set out in Manual of BBL.RTM. Products and Laboratory Procedures, 4th ed., D. A. Power and P. J. McCuen (eds.), 1988. All chromogens were added aseptically to the base post autoclaving at a concentration of 0.1 g/l of media. Chromogens were either added as a powder (if water soluble) or predissolved in DMSO. In one instance, chromogens were added before autoclaving.
The chromogenic substrates used are set forth below:
In construing the chart below:
X=5-bromo-4-chloro-3-indolyl
Mag=5-bromo-6-chloro-3-indolyl
Sal=6-chloro-3-indolyl
______________________________________Chromogenic Substrate Abbr.______________________________________X-N-Acetyl-.beta.-D-galactosaminide X-AcglmnMagenta-caprylate M-CapX-galactopyranoside (X-gal) + IPTG X-Gal + IPTGX-galactopyranoside X-GalX-glucoside X-GlucosideSal-glucuronide Sal-GlucuroMagenta-phosphate Mag-PhosX-Sulfate X-SulfX-Glucuronide X-GlucuroMix (Sal-Glucuro, Mag-phos, X-Acglmn, BG-Gal) MixTSA II 5% SB - no chromogens TSA cont.______________________________________
A total of 55 strains (abbreviations as defined in Table V) were tested using the above media. Plates were incubated in CO.sub.2 and air, or CO.sub.2 only. All plates were read at 18-24 h for color, hemolytic activity, and growth as compared to the non-chromogenic control.
Colors were evaluated using Pantone Equivalents in order to more precisely and consistently identify the resulting colors, as noted below
______________________________________Color Pantone Equivalent______________________________________ b = blue 17-4336 .fwdarw. 19-4340gb = grey blue 17-4412 p = purple 18-3513gp = grey purple 15-3807pb = purple blue 18-3932NG = no growth______________________________________
Used in front of the color abbreviations noted above, d=dark; l=light; vl=very light.
The bacterial strains were prepared in normal saline at 0.5 McFarland and diluted 1 in 10.
The plates were inoculated using the standard streak plate method or using a Steers replicator. Steers, E. et al., Antibiot. Chemother., 9:307-311 (1959).
Results are tabulated below in Tables V and VI.
TABLE V__________________________________________________________________________Colony Color on Various Chromogenic TSA II with 5% Sheep Blood(Summary) No. ChromogensStrain Abbrev. Tested X-Acglmn X-Gal X-Glucoside X-Glucuro Mag-phos M-Cap X-Sulf__________________________________________________________________________Aeromonas hydrophilia aehy 3 + 0 v 0 + 0 0Branhamella catarrhalis brca 2 0 0 0 0 v 0 0Candida albicans caal 2 + 0 0 0 0 0 0Citrobacter freundii cifr 2 0 + 0 0 0 0 +Entercoccus faecalis enfa 6 + v + 0 + 0 0E. faecium enfu 1 + + + 0 + 0 0E. coli (non-0157) esco 3 0 + 0 + v 0 0Klebsiella pneumoniae klpn 3 0 + + 0 v 0 0Pseudomonas aeruginosa psae 3 0 0 v 0 0 0 0Salmonella spp. sasp 2 0 0 0 0 + 0 0Serratia marcescens sema 1 + + + 0 0 0 0Streptococcus agalactiae stag 3 0 0 + 0 + 0 0Staphylococcus aureus stau 5 0 0 0 0 v 0 0Staphylococcus Coag Neg step 5 v v 0 0 v 0 0S. milleri stmi 2 0, <grth 0 0 0 no growth 0 0S. pneumoniae stpn 6 0, <grth 0 v 0 0, 0 0 <growthS pyogenes stpy 6 0, <grth 0 v v +, 0 0 <grth 55__________________________________________________________________________ + = color detected v = color varied slightly with strain 0 = no color detected <= decreased growth vs. control
TABLE VI__________________________________________________________________________Colony Color on Various Chromogenic TSA II with 5% Sheep Blood(Detail) Refer- Incub Incub X-Gluc- X- Sal- X-Strain ence No. atm time X-Acglm M-Cap Xgal/IPTG X-Gal oside Glucuro Glucuro Mag-Phos Sulf Mix TSA__________________________________________________________________________ controlaehy 7965 CO.sub.2 21 h gb 0 g gb 0 NT 0 gp 0 pb l yellowaehy 7966 CO.sub.2 21 h gb 0 0 gb g NT 0 gp 0 pb yellowaehy 49847 CO.sub.2 21 h gb 0 lg gb gb NT 0 g 0 pb yellowbrca 25238 CO.sub.2 21 h 0 0 0 0 0 NT 0 0 0 0 greybrca 25240 CO.sub.2 21 h 0 0 0 0 0 NT 0 p 0 0 l greycaal 10231 CO.sub.2 24 h gb 0 0 0 0 NT 0 0 0 gb greycakr 34135 CO.sub.2 21 h 0 0 0 0 0 NT 0 lgp 0 0 greycifr 8454 CO.sub.2 21 h 0 0 gb gb 0 NT 0 0 0 0 greycifr 33128 CO.sub.2 21 h 0 0 gb gb 0 NT 0 0 gb gp greyenfa 597 CO.sub.2 21 h db 0 0 0 b NT 0 lp 0 db greyenfa 10741 CO.sub.2 21 h db 0 lgb gb db NT lp 0 db greyenfa 12953 CO.sub.2 21 h db 0 0 0 db NT 0 lp 0 b greyenfa 14506 CO.sub.2 21 h db 0 0 lgb db NT 0 lp 0 db greyenfa 29212 CO.sub.2 21 h db 0 gb gb db NT 0 0 db greyenfa 29212 CO.sub.2 24 h db 0 lgb vlgb b NT 0 p 0 db greyenfu 49032 CO.sub.2 21 h db 0 0 gb b NT 0 vlgp 0 vlgp greyesco 11775 CO.sub.2 21 h 0 0 b b 0 NT 0 lp 0 gp greyesco 25922 CO.sub.2 24 h 0 0 b b 0 NT 0 lgb 0 b greyesco 33605 CO.sub.2 21 h 0 0 gb gb 0 NT 0 0 0 gp greyklpn 13883 CO.sub.2 21 h 0 0 gb gb gb NT 0 0 0 lp grey (mucoid)klpn 33495 CO.sub.2 24 h 0 0 b b b NT 0 p 0 b greyklpn 33606 CO.sub.2 21 h 0 0 gb gb gb NT 0 0 0 lp greymilu 9341 CO.sub.2 21 h 0 0 0 0 0 NT 0 NG 0 0 l yellowpsae 9027 CO.sub.2 21 h 0 0 0 0 0 NT 0 0 0 0 greypsae 15442 CO.sub.2 21 h 0 0 0 0 0 NT 0 0 0 0 yellowpsae 33607 CO.sub.2 21 h 0 0 0 0 lgb NT 0 0 0 0 l yellowsaty 19430 CO.sub.2 21 h 0 0 0 0 0 NT 0 p 0 p greysatym 14028 CO.sub.2 21 h 0 0 0 0 0 NT 0 p 0 p greysema 13880 CO.sub.2 21 h gb 0 gb gb gb NT 0 0 0 gb greystag 4768 CO.sub.2 21 h 0 0 0 0 b NT 0 lp 0 lp greystag 12386 CO.sub.2 24 h 0 0 0 0 b NT gp lp 0 p whitestag 12386 CO.sub.2 21 h 0 0 0 0 b NT 0 p 0 p white (beta)stau 25923 CO.sub.2 24 h 0 0 0 0 0 NT 0 lp 0 lp whitestau 1006 CO.sub.2 21 h 0 0 0 0 0 NT 0 0 0 0 white (beta)stau 1007 CO.sub.2 21 h 0 0 0 0 0 NT 0 0 0 0 white (beta)stau 1008 CO.sub.2 21 h 0 0 0 0 0 NT 0 vlp 0 vlp whitestau 1009 CO.sub.2 21 h 0 0 0 0 0 NT 0 vlp 0 vlp white (beta)step 12228 CO.sub.2 21 h 0 0 0 0 0 NT 0 0 0 vlp whitestep cdc1 CO.sub.2 21 h 0 0 0 0 0 NT 0 lp 0 lp greystep cdc2 CO.sub.2 21 h 0 0 0 0 0 NT 0 0 0 0 whitestep cdc3 CO.sub.2 21 h 0 0 b b 0 NT 0 0 0 0 whitestep cdc4 CO.sub.2 21 h 0 0 0 0 0 NT 0 lp 0 0 greystmi 6249 CO.sub.2 21 h NG 0 0 0 0 NT 0 NG NG NG NGstpn 6303 CO.sub.2 24 h 0 0 0 0 0 NT 0 0 0 0 alphastpn 6305 CO.sub.2 24 h 0 0 0 0 0 NT 0 0 0 0 alphastpn 1259 CO.sub.2 21 h 0 0 0 0 0 NT 0 0 0 0 alpha (mucoid)stpn 1260 CO.sub.2 21 h 0 0 0 0 0 NT 0 0 0 NG alphastpn 1331 CO.sub.2 21 h 0 0 0 NG NG NT NG NG NG NG NGstpn 1628 CO.sub.2 21 h 0 0 0 0 0 NT 0 0 0 0 alphastpy 19615 CO.sub.2 21 h 0 0 0 0 gb NT 0 lp 0 lp white (beta)stpy 49117 CO.sub.2 21 h 0 0 0 0 0 NT 0 lp 0 lp white (beta)stpy 51339 CO.sub.2 21 h 0 0 0 0 0 NT 0 lp 0 lp white (beta)stpy 1027 CO.sub.2 21 h 0 0 0 0 0 NT 0 vlp 0 vlp NGstpy 1028 CO.sub.2 21 h 0 0 0 0 lgb NT 0 vlp 0 vlp grey (beta)stpy 1029 CO.sub.2 21 h 0 0 0 0 lgb NT 0 vlp 0 vlp NGstpy 1030 CO.sub.2 21 h 0 0 0 0 gb NT 0 vlp 0 vlp grey (beta)caal 10231 air 24 h 0 0 0 0 0 NT 0 0 0 0 grey (darker)enfa 597 air 21 h b 0 0 0 b NT 0 lp 0 db greyenfa 10741 air 21 h b 0 lb lgb b NT 0 p 0 db greyenfa 12953 air 21 h b 0 0 0 b NT 0 p 0 db greyenfa 14506 air 21 h b 0 0 0 b NT 0 p 0 db greyenfa 29212 air 21 h b 0 lgb gb b NT 0 p 0 db greyenfa 29212 air 24 h db 0 lgb vlgb b NT 0 p 0 db greyenfu 49032 air 21 h lb 0 0 db b NT 0 lgp 0 lp grey (gb48h)esco 25922 air 24 h 0 0 b b 0 NT 0 lgb 0 b greyklpn 33495 air 24 h 0 0 b b b NT 0 p 0 b greystag 4768 air 21 h 0 0 0 0 lb NT 0 p 0 lp greystag 12386 air 24 h 0 0 0 0 b NT gp lp 0 p greystau 25923 air 24 h 0 0 0 0 0 NT 0 lp 0 lp whitestep 12228 air 21 h 0 0 0 0 0 NT 0 lp 0 0 white (p-48h)step cdc1 air 21 h 0 0 0 0 0 NT 0 lp 0 lp greystep cdc2 air 21 h 0 0 0 0 0 NT 0 0 0 0 whitestep cdc3 air 21 h lb 0 b b 0 NT 0 0 0 0 whitestep cdc4 air 21 h 0 0 0 0 0 NT 0 lp 0 vlp greystmi 6249 air 21 h 0 0 0 0 0 NT 0 0 0 NG NGstpn 6303 air 24 h 0 0 0 0 0 NT 0 db 0 0 alphastpn 6305 air 24 h 0 0 0 0 0 NT 0 0 0 0 alphastpy 19615 air 21 h 0 0 0 0 lgb NT 0 lp 0 lgb white (beta) (p48h)stpy 49117 air 21 h 0 0 0 0 0 NT 0 p 0 lp white (beta)stpy 51339 air 21 h 0 0 0 0 lgb NT 0 p 0 lp white (beta)aehy 7965 CO.sub.2 18 h db NT NT NT 0 0 NT NT NT NT grey hemaehy 7966 CO.sub.2 18 h db NT NT NT 0 0 NT NT NT NT grey hemaehy 49847 CO.sub.2 18 h db NT NT NT gb 0 NT NT NT NT grygrn hembrca 25238 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT greybrca 25240 CO.sub.2 18 h NG NT NT NT 0 0 NT NT NT NT greycaal 10231 CO.sub.2 18 h b NT NT NT 0 0 NT NT NT NT greycakr 34135 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT greycifr 8454 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT dark greycifr 33128 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT greyenfa 597 CO.sub.2 18 h db NT NT NT db 0 NT NT NT NT greyenfa 10741 CO.sub.2 18 h db NT NT NT db 0 NT NT NT NT greyenfa 12953 CO.sub.2 18 h db NT NT NT db 0 NT NT NT NT greyenfa 14506 CO.sub.2 18 h db NW NT NT db 0 NT NT NT NT greyenfa 29212 CO.sub.2 18 h db NT NT NT db 0 NT NT NT NT greyenfu 49032 CO.sub.2 18 h lb NT NT NT db 0 NT NT NT NT greyesco 11775 CO.sub.2 18 h 0 NT NT NT 0 b NT NT NT NT greyesco 33605 CO.sub.2 18 h 0 NT NT NT 0 b NT NT NT NT grey hemklpn 13883 CO.sub.2 18 h 0 NT NT NT b 0 NT NT NT NT white greyklpn 33495 CO.sub.2 18 h vlgb NT NT NT gb 0 NT NT NT NT white greyklpn 33606 CO.sub.2 18 h 0 NT NT NT gb 0 NT NT NT NT white greymiiu 9341 CO.sub.2 18 h ly NT NT NT ly ly NT NT NT NT lt yellowpsae 9027 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT dark greypsae 15442 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT dark greypsae 33607 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT dark greysaty 19430 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT greysatym 14028 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT dark greysema 13880 CO.sub.2 18 h gb NT NT NT bg 0 NT NT NT NT dark greystau 25923 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT whitestau t006 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT whitestau t007 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT whitestau t008 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT whitestau t009 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT whitestep 12228 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT whitestep cdc1 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT whitestha cdc2 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT whitestsa cdc3 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT whitestep cdc4 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT white betastmi 6242 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT alphastpn t259 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT alphastpn t260 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT alphastpn t331 CO.sub.2 18 h drkr<muc NT NT NT 0 0 NT NT NT NT alpha mucstpn t628 CO.sub.2 18 h 0 NT NT NT bg 0 NT NT NT NT alphastpy 51339 CO.sub.2 18 h 0 NT NT NT 0 db beta NT NT NT NT betastpy t027 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT betastpy t028 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT betastpy t029 CO.sub.2 18 h 0 NT NT NT 0 0 NT NT NT NT betastpy t030 CO.sub.2 18 h 0 NT NT NT lgb 0 NT Nt NT NT beta__________________________________________________________________________ Strain Abbreviation as defined in Table V NG = Growth NT = Not tested 0 = No Color alpha = alpha hemolysis beta = beta hemolysis
Again, it was surprising to discover that chromogenic substrates applied to the surface of, or incorporated within a highly colored medium (i.e., a nutrient medium containing blood or hemin) could help to identify and/or differentiate microorganisms.
As will be observed from review of the data tabulated above, Group B Strep (3 of 3 strains) gave blue colonies on X-glucoside TSA II. Colony hemolytic activity was slightly reduced. The CAMP test works well on X-glucoside TSA II (as described in the Manual of BBL.RTM. Products and Laboratory Procedures). PYR test (as described in Manual of Clinical Microbiology, 6th ed., P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 1995 ASM Press, Washington, D.C.) would be needed to rule out S. pyogenes or Group D strep which may also be blue. 4 of 7 S. pyogenes gave light grey blue color. 7 of 7 Enterococcus gave a blue color.
Group D Strep (7 of 7 strains) gave dark blue colonies on X-Acglmn. A few Gram Negative strains, one C. albicans and one S. epidermidis also gave blue colonies.
CO.sub.2 incubations typically gave slightly stronger color reactions vs. air incubation for all chromogens.
Addition of the X-chromogens prior to autoclaving did not effect performance.
M-Cap and X-Sulf were inactive for most organisms. Literature suggests that sodium desoxycholate may be important for the M-Cap reaction with Salmonella spp. One strain of Citrobacter gave a grey blue with X-Sulf TSA II.
Some of the chromogens, especially magenta substrates, caused the blood agar to darken after incubation, particularly after CO.sub.2 incubation. This darkening of the plate reduced the readability of the purple color reactions and hemolytic reactions.
X-gal was not particularly useful for Gram-positives. IPTG appeared to reduce the reactivity for some strains with X-gal. E. faecium (ATCC 49032) gave dark blue on X-Gal but no color on X-Gal with IPTG.
As would be expected, chromogens that produced colony colors that clearly contrast with the red surface of the blood plate are optimal. The blue-chromogen, X-substrate (5-bromo-4-chloro-3-indoxyl) has an absorption max (nm) of 615 which is most differentiated from the red blood plates color of approx. 515 nm. Some chromogens which produce purple/red/magenta colonies are not as easy to read against the red blood plate background.
Based on the above data, the color contrast can be summarized as follows:
______________________________________ Absorption Contrast onChromogen Abbr. Colony max (nm) Blood Plate______________________________________5-Bromo-4-chloro- X- blue 615 Good3-indoxyl5-Bromo-6-chloro- Mag- purple/red 565 Fair/poor3-indoxyl6-chloro-3-indoxyl Sal- grey purple 540 Poor*______________________________________ *Compound unstable, gave poor reactions with positive control (E. coli 25922)
While the examples use TSASB and chocolate agar as the blood or hemin containing culture media, the invention is not limited to these two media.
It is expected that other, similar blood or hemin containing media, such as Columbia Agar Base, with the addition of blood, which is widely used in Europe, would also be useful in the present invention. This media is commercially available, and contains more amino acid and carbohydrate than TSASB.
It is noted, however, that limited efforts to use commercially available Brucella Agar with blood, produced bacterial growth but no color change.
The above Examples are intended to be purely exemplary, and are not intended to in any way limit the scope of the present invention.

Method for differentiating microorganisms in a sample

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Ashdown, L.. R.; "Pathology"; vol. 11(2), pp. 293-297, Feb. 1979.
Wu, William, G.; "Medical Microbiology"; 2nd Edition, pp. 349, 1989.