Chapter 14 - The enteric bacteria (the family <i>Enterobacteriaceae</i>)

14 - 1 Introduction

The term enteric bacteria (or enterics) is generally used in reference to organisms of the Family Enterobacteriaceae, many members of which occur in the enteric tract of humans and animals in health and disease. Other residents of the intestinal tract (e.g., Clostridium, Bacteroides and various lactobacilli and streptococci) also might be called enterics, but convention reserves the term for the Family Enterobacteriaceae. All members of this family possess these characteristics in common:

• gram-negative
• rod-shaped
• facultatively anaerobic
• positive for true catalase and cytochromes
• ferment glucose by one of two major pathways to a variety of end products
• oxidase-negative
• possess the enterobacterial common antigen in the cell wall

Enterics can be found in a variety of natural habitats, not just in the intestinal tract. Selective isolation of these organisms is assisted by the use of media containing agents which inhibit gram-positive organisms. MacConkey Agar is a popular selective medium, although this medium can also support the growth of many gram-negative organisms other than the enterics. (Neisseria is one of a number of gram-negative bacteria which cannot grow on MacConkey Agar.) There is no one medium on which all enterics will grow with the exclusion of all other organisms.

Most enterics are motile by peritrichous flagella; two major exceptions are Klebsiella and Shigella. Most will reduce nitrate to nitrite but never to nitrogen gas. Depending on the specific organism, various sugars may be fermented and/or respired (yielding energy - see Exp. 5.1), and one or more amino acids (such as lysine, ornithine, arginine, glutamic acid and histidine) may be decarboxylated. Fermentation and decarboxylation are anaerobic processes and will result in acid and alkaline reactions, respectively. Another anaerobic process - production of hydrogen sulfide from thiosulfate - is possessed by some of the enterics.

For any organism in the enteric family, glucose (and other carbohydrates, depending on the organism) is fermented to pyruvic acid and beyond by either the mixed-acid or butanediol pathways. End-products of the mixed-acid type of fermentation include lactic, acetic, succinic and formic acids and ethanol. A very low pH is rapidly achieved and maintained. After incubation in a standardized medium (MR-VP Broth), the addition of methyl red (a pH indicator) results in a red color (a positive reaction), an indication of pH 4.4 or below. Organisms possessing the butanediol type of fermentation produce the same end products and low pH by one day of incubation but neutral products (acetoin and/or 2,3-butanediol) and carbon dioxide are then formed at the expense of one or more of the acids. After an additional day of incubation, the pH is at 6.2 or above where the addition of methyl red results in a yellow color. The Voges-Proskauer (VP) test indicates the presence of neutral products directly. Therefore, with rare exceptions, an MR-positive organism will be VP-negative, and an MR-negative organism will be VP-positive.

Many enterics produce gas during carbohydrate fermentation which collects in a Durham tube. This gas is the result of the enzyme formic hydrogenlyase which splits formic acid into hydrogen and carbon dioxide.

As expected for most chemoheterotrophic bacteria, ammonia is produced from aerobic deamination of one or more amino acids such as those found in peptones and yeast extract. The combination of this alkaline product with acids produced from carbohydrate fermentation produce a net reaction which is important in the interpretation of some differential media such as Kligler Iron Agar. As for many carbohydrates, amino acids may be utilized as sources of carbon and energy (the latter via respiration).

Thus it may be seen that an unknown isolate may be identified to the enteric family and ultimately to genus and species by the application of the appropriate tests, many of which are used in this exercise. Certain other gram-negative rods may resemble the enterics superficially. For example, Pseudomonas shares many habitats with enterics and is often found on the plating media utilized in enteric isolation, but it can be easily recognized by its strictly aerobic nature, its failure to ferment any sugar and its (usually) oxidase-positive reaction. Vibrio, Aeromonas and the luminescent (light-producing) genus Photobacterium, all of which are facultatively anaerobic, appear even more closely related to the enterics, but they are distinguished easily by the oxidase test.

Selected Genera of the Family Enterobacteriaceae

Escherichia, Enterobacter and Klebsiella: These organisms are often considered together, as most strains of these genera are distinguished easily by their ability to ferment lactose rapidly to acid and gas. These strains are the true coliforms, useful as indicators of water quality (see Exercise 15). Escherichia coli is the predominant facultatively anaerobic organism in the large intestine, and its presence in water, food or anywhere indicates fecal contamination and the possibility of associated intestinal pathogens. E. coli may cause diseases of the urinary tract, and certain strains cause severe intestinal disease. The various species of Enterobacter and Klebsiella are widely distributed on plants and in soil and are often found in the gastrointestinal tract. The finding of these organisms in drinking water indicates surface soil contamination. K. pneumoniae is an occasional cause of pneumonia in humans.

Genetically, Shigella and E. coli are virtually identical. Were they to be discovered and named today, they would be grouped into one species. However, Shigella has been set apart traditionally for its consistent non-motility, its failure to ferment lactose and its cause of bacillary dysentery, characterized by severe abdominal pain and bloody diarrhea. Only a few ingested cells are needed to result in disease.

Citrobacter is a commonly-found non-pathogenic enteric. In food and medical laboratories, colonies of typical strains (those which are lactose-negative and H₂S-positive) appear identical to those of Salmonella on most isolation media. Subsequent biochemical testing will differentiate these genera easily. Certain other strains of Citrobacter which can ferment lactose rapidly to acid and gas are thus considered coliforms and are occasionally isolated in Experiment 15.

Salmonella is a genus of pathogenic organisms infecting humans and many mammals, birds and reptiles. Organisms in this genus are so genetically similar that they are now considered as belonging to one or two species. Subdivision of the genus has been made to at least seven genetically-distinct subgroups (subspecies) and further to well over 2000 serovars (formerly called serotypes). Each serovar is distinguished by a unique combination of cell wall (O) and flagellar (H) antigens (see Exp. 14.2). Serovar recognition is important in epidemiology. Often an outbreak or epidemic caused by strains of one serovar can be traced to a common source. Decades ago, it was the practice to consider each serovar a species. Now, most serovars are designated for convenience with names which are written like species names. The three serovars Salmonella typhimurium, S. enteritidis and S. heidelberg are responsible for about half of the cases of human Salmonella infection in the United States.

Most serovars of Salmonella cause gastroenteritis of varying degrees of severity, with or without bacteremia. The source of gastroenteritis is usually contaminated food containing over 106 cells/g (or ml). Symptoms generally appear in 8 to 30 hours after ingestion and include nausea, fever, diarrhea, and abdominal pain, usually subsiding in one to two days. Certain host-adapted, biochemically-distinct serovars (which may be termed bioserovars) cause life-threatening illnesses such as typhoid fever by S. typhi, hog cholera by S. choleraesuis (also pathogenic to humans) and fowl typhoid by S. gallinarum.

Organisms of the genus Edwardsiella are known to cause disease in humans and a variety of warm and cold-blooded vertebrates. E. tarda is an occasional opportunistic pathogen for humans, causing wound infections and occasional gastroenteritis. E. tarda and E. ictaluri have caused massive infections of commercially-raised catfish with considerable economic loss.

Yersinia includes the species Y. pestis, the causative agent of bubonic plague. Other diseases produced by Yersinia include a severe gastroenteritis in humans and red-mouth disease in trout and salmon.

The Proteus group of enterics (Proteus, Providencia and Morganella) is easily distinguished by the ability to deaminate the amino acid phenylalanine. These organisms are relatively non-pathogenic but are capable of causing urinary tract infections and occasional gastroenteritis. Fecal matter, sewage and soil, especially where animal protein is decomposing, are common sources for these organisms. Scombroid food poisoning, caused by the decarboxylation by Morganella morganii of histidine to histamine (which causes severe allergy-like symptoms), is associated with certain fish of high histidine content such as tuna and mackerel.

Erwinia is a genus of plant pathogens which produce a variety of diseases including wilts and soft rots. Erwinia has been implicated in cases of septicemia caused by contaminated intravenous fluids.

Serratia is a widespread organism found in soil, water and clinical material. Some strains produce a bright, red pigment (prodigiosin) as was seen in Experiment 6. Serratia tends to hydrolyze proteins, nucleic acids and chitin rapidly. Some insect diseases and cases of human pneumonia are caused by Serratia.

Many additional genera are also included in the family Enterobacteriaceae which now contains about thirty genera and well over a hundred species! The luminescent organism Photorhabdus is found frequently in association with nematodes. Hafnia and Obesumbacterium are occasional brewery contaminants. Others are mainly of obscure clinical or environmental interest and are rarely isolated. In the mid 1990's, an unusual group of strains (the G30 Biogroup) isolated from six Wisconsin lakes was discovered at the U.W. Department of Bacteriology and was shown by the Centers for Disease Control to be a distinct enteric genus based on genotypic and phenotypic studies. It is presently established as CDC Enteric Group 121. (You may often see as yet unnamed enteric groups referred to when you read about clinical organisms.) Upon formal publication the designation will be Aquamonas haywardensis (water unit from Hayward).

Overview of isolation and identification (with reference to our procedure).

The principles of enrichment and isolation continue to apply to the enterics. For example, in clinical and food microbiology laboratories, where enteric pathogens such as Salmonella and Shigella are implicated in a disease or a contaminated food, the procedure may be outlined as follows:

1. Obtain specimen. If the specimen is expected to contain a large, active population of the suspected organism (such as a diarrhea specimen from an individual suffering from an intestinal infection), one may go directly to step 3.
2. Inoculate specimen into selective enrichment broth medium (or media) such that the organisms to be looked for are given every opportunity to grow with minimum interference from the growth of other organisms. The selective agents included (also in the following plate media) are primarily intended to inhibit gram-positive organisms. Depending on the amounts and types of selective agents included, some highly-selective media may inhibit certain gram-negative organisms (including some enterics) as well.
3. Streak onto selective-differential plate media for isolated colonies. Once these are obtained, careful selection is made of colonies to be picked for further analysis.
4. One must pick only from the very top of the colonies. Contaminating organisms on the surface of the medium may be present. Though inhibited, they are still viable! If in doubt about purity, one can streak onto a non-selective medium.
5. Typical strains of Salmonella and Shigella do not ferment lactose or sucrose. Therefore, if either of these organisms is suspected, one may simply pick non-acid-producing colonies off media which contain these sugars. However, many other gram-negative rods do not ferment lactose or sucrose including Pseudomonas which does not ferment anything. Also, some exceptional strains of Salmonella are lactose-positive. Anticipating this probability, a supplementary medium is used which does not differentiate according to fermentation reactions (Bismuth Sulfite Agar).
6. Inoculate preliminary biochemical screening media from the chosen colonies. One has no way of identifying specific organisms on the plates. Also, one would not wish to waste time and money by inoculating all of the media necessary for identification from each chosen colony! Therefore, one or more media such as Triple Sugar Iron Agar (or Kligler Iron Agar), Lysine Iron Agar and (sometimes) Motility Indole Ornithine Medium are inoculated from each colony. Each of these media yields several useful pieces of information about the colony picked. With any of these media, non-fermenting organisms such as Pseudomonas are recognized and discarded, thus saving time and money in the coming procedures.
7. Perform serological tests. As explained in the introduction, serovars of Salmonella are distinguished by the identification of their O and H antigens, and a suspected culture of Salmonella may be tested with antibodies, singly and in combinations, for identification to whatever level is desired: genus, serological group or serovar. Serological identification of Shigella and certain other enterics may also be attempted.
8. Perform confirmatory biochemical tests. Various differential media can be inoculated from one of the preliminary screening media.

In Experiment 14.1, an abbreviated version of the above will be performed on a mixed unknown consisting of three organisms. Two of the three organisms are enterics to be identified. The third is a strain of Pseudomonas which will be recognized sooner or later as a non-enteric and then discarded during or before the third period. We will forego any selective enrichment broth media and streak the mixture directly onto the plates for isolated colonies. In Experiment 14.2 we will perform a serological procedure to identify a strain of Salmonella to a serological group, but we will not be doing this for any of the unknowns. There are no enteric pathogens among the unknowns. Make sure nothing is identified ultimately as Salmonella or Shigella. Refer to the appendices at the end of Experiment 14 concerning the various media and organisms.

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14 - 2 Isolation and identification of an unknown enteric

Period 1

Materials

Saline suspension of two enterics (differing in abilities to ferment lactose and/or produce hydrogen sulfide) plus a non-enteric (Pseudomonas)

2 plates of Modified MacConkey Agar

Optionally, plates of Brilliant Green Agar and/or XLD Agar also may be available.

Record the number of your unknown.

1. Streak the mixed unknown onto each plate. Be sure to streak for isolated colonies!
2. Incubate the plates at 37°C. If the next period is two or more days away, bring the plates to the stage for incubation.)

Period 2

Materials

3 or more slants of Kligler Iron Agar (KIA)

(Optionally, slants of Lysine Iron Agar (LIA) may also be available.)

Demonstrations of various enterics on selective-differential plating media

1. Observe the demonstrations of some enterics Figure 14-1 (including Salmonella and Shigella) on various plating media used in the isolation of enterics. The instructor will provide explanatory material.
2. Examine your streak plates carefully, Figure 14-2. Note the characteristics and colors associated with only the well-isolated colonies. The three different organisms should be easy to differentiate on the Modified MacConkey Agar. Label each of the different chosen colonies by number or letter and record in your notes the appearance of each. (Also, indicate the specific medium of isolation if media in addition to MacConkey Agar were used.)

(1)How is a non-lactose-fermenting organism able to grow on MacConkey Agar?

(2)At this point, would you expect to be able to differentiate between enterics and other organisms (such as Pseudomonas) on the plates? Why or why not?

3. Inoculate each chosen colony into the same-numbered tube of KIA by the use of the needle as follows: First, stab the "butt" of the medium all the way to the bottom and then streak along the top of the slanted portion from bottom to top. (If LIA is also used, the medium is inoculated in like manner.)
4. If the next period is more than one day away, place the KIA tubes in the baskets on the stage.

The tubes will be refrigerated and then placed in the 37°C incubator a day before the next period. For the proper interpretation of KIA, it is very important that the reactions in the tubes be recorded for just one day of incubation.

Period 3

Materials

2 slants of Phenylalanine Agar

2 tubes of Lactose Fermentation Broth

2 tubes of MR-VP Broth

2 tubes of Motility Indole Ornithine (MIO) Medium

2 slants of Simmons Citrate Agar

2 tubes of Lysine Decarboxylase Broth

2 tubes of Decarboxylase Control Broth

4 tubes of sterile mineral oil

Demonstration of KIA reactions

1. Observe the demonstration of reactions in KIA, Figure 14-3. Note how Salmonella and Shigella differ from each other and how they are similar to certain other enterics in the medium.
2. Observe your tubes of KIA. Discard the tube for any isolate which is negative for glucose fermentation (red slant and orange butt). This is the non-enteric (Pseudomonas) to which you need not pay any more attention. The remaining tubes should differ in the indication for lactose fermentation (yellow slant if positive) and/or H₂S production (black color throughout the "butt" if positive). Retain two clearly-different tubes (discarding the rest, if any) and record the reactions carefully. These two slant cultures are your enteric isolates to identify and will provide the inocula for the next step.
3. From each tube of KIA, inoculate each of the media listed under Materials, above. Carefully stab-inoculate (with the needle) the tube of MIO and do not mix! All of the other media should be inoculated in "normal" fashion; do not stab-inoculate the slants.
4. Overlay each inoculated tube of Lysine Decarboxylase Broth and Decarboxylase Control Broth with sterile mineral oil as you did in Experiment 4-3 for Glucose O/F Medium.

What is the principle behind the development of anaerobic conditions in media overlayed with mineral oil? Consider the oxygen relationship of these organisms. (Recall what was done in setting up the enrichment for photosynthetic bacteria)

5. Incubate the tubes as follows:
• Unless you are coming into lab tomorrow, bring the MIO and Phenylalanine Agar to the front of the lab for one-day incubation (immediate refrigeration followed by incubation at 37°C for one day prior to the next period).
• Place all of the other tubes in the 37°C incubator. These media may be examined at any time after one day of incubation except for the MR-VP Broth which must be incubated for at least two days before any tests are made.

Period 4

Materials

Dropper bottles of FeCl₃, methyl red and Kovacs reagent

Be sure to consult with your neighbors such that positive and negative reactions can be seen for each of the media.

1. Phenylalanine Agar. Add about one-half dropperful of the FeCl₃ solution. If deamination of phenylalanine has taken place, the reagent will react with the phenylpyruvic acid formed, and a dark green color will appear in the slant region. Figure 14-4 shows + and - reactions for phenylalanine agar.
2. Lactose Fermentation Broth. Any degree of yellow color indicates fermentation. How does the result compare with what was seen in KIA and the original isolation medium? Figure 14-5 shows + and - reactions for Lactose Fermentation Broth
• A slight amount of yellow color with little or no gas should be recorded as a weakly-positive reaction. Such organisms probably would have appeared lactose-negative on MacConkey Agar and KIA.
• A whitish color in the bottom half of the tube is due to reduction of the pH indicator and is not an indication of acid.
3. MR-VP Broth. (Be sure that this medium was incubated for at least two days.) Add a half-dropperful of the methyl red reagent to the tube. Do not mix; just let the reagent form a layer at the top of the medium. Figure 14-6 shows the + and - methyl red reactions
• Red color: Positive reaction, indicative of mixed-acid fermentation; pH has dropped and remained at or below 4.4.
• Yellow color: Negative reaction, indicative of butanediol fermentation; pH has dropped and then risen to 6.2 or above.
• Orange color: Record as an "equivocal" reaction.
4. The Voges-Proskauer (VP) Test: (This test is not run routinely in this course, but the procedure is given here for those interested or in case it is done optionally.) A second tube of MR-VP Broth is inoculated and incubated. Then, 12 drops of alpha-naphthol reagent and 4 drops of 40% KOH are added. Results are noted after 10-30 minutes:
• Red color: Positive reaction for the presence of neutral products (acetoin and/or 2,3-butanediol), indicative of butanediol fermentation. Such organisms are usually methyl red-negative.
• Yellowish color: Negative reaction, indicative of mixed-acid fermentation. Such organisms are usually methyl red-positive.
5. Motility Indole Ornithine (MIO) Medium.
6. MIO - Motility. Observe for cloudiness in the medium (growth away from the stab line). For a non-motile organism, growth may be seen along cracks in the medium caused by gas production, but there will be clear pockets of no growth. Figure 14-8 shows various reactions for MIO medium.
7. MIO - Ornithine Decarboxylation. Observe the lower three-quarters (anaerobic region) of the medium for change in color of the pH indicator; growth must be present in this part of the tube.
• Gray, blue or purple color: Positive reaction for ornithine decarboxylation - formation of a highly alkaline product, overneutralizing the acid produced from glucose fermentation.
• Yellow color: Negative reaction. Yellow color is due to acid production from glucose fermentation.
8. MIO - Indole Production. As for the Tryptone Broth (Exp. 7), add about one-half dropperful of Kovacs reagent to the medium. A red ring indicates production of indole from the breakdown of tryptophan.
9. Simmons Citrate Agar, Figure 14-9. If utilization of citrate (the sole carbon and energy source in the medium) has taken place, an alkaline reaction results. The pH indicator turns bright blue in the slant region.
10. Decarboxylase Control Broth, Figure 14-10. This medium is identical to the following medium except that that lysine is not included. All enterics should be able to grow in this anaerobic medium, fermenting the glucose and producing acid, resulting in a yellow color. A strict aerobe such as Pseudomonas will not grow, in which case the medium will remain somewhat purple in color.
11. Lysine Decarboxylase Broth Figure 14-10. (If growth and acid production are not seen in the control broth, results in this medium are meaningless.)
• Gray, blue or purple color (a color "darker" than that of the corresponding control): Positive reaction for lysine decarboxylation - formation of a highly alkaline product, overneutralizing the acid produced from glucose fermentation.
• Yellow color (identical to the corresponding control): Negative reaction.

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14 - 3 Serological identification of a <i>Salmonella</i> culture

In the serological identification of a suspected Salmonella isolate, one utilizes antibodies pre-pared against specific antigens found on the cell wall and flagella. Blood serum containing one or more known antibodies is called antiserum. Agglutination (defined as a clumping of particles - in this case, bacterial cells) is observed where there is a match between antibodies in the antiserum and the homologous antigens on the particles.

For the detection of specific cell wall ("O") antigens, cells are obtained from a solid medium such as Triple Sugar Iron Agar or Kligler Iron Agar. Slide agglutination tests are performed with indi-vidual antisera as demonstrated in our procedure below. For the detection of flagellar ("H") antigens, the tube agglutination test is performed: BHI Broth cultures of the isolate are mixed with antisera, and an antibody-antigen match results in an agglutination of cells which settles rather quickly out of suspension.

To identify a Salmonella isolate with specificity - especially in the interests of epidemiology - one desires an identification more precise than a species name. In fact, species names are usually ignored. In the slide agglutination test, a reaction with antibodies unique to a serological group will lead to application of the group designation for the isolate as follows: Organisms which possess O antigen 2 are designated Salmonella Group A, those with O antigen 4 are designated Salmonella Group B, those with O antigens 6 and 7 are designated Salmonella Group C1, and so on.

With specific antisera, several cell wall and flagellar antigens can be detected for a Salmonella isolate, and identification becomes more precise. Based on the results of the slide and tube agglutination tests, the isolate is given an antigenic formula such as the following: 30:i:e,n,z₁₅ With this format, the numbers given before the first colon (:) represent specific O antigens, the next set of numbers represent flagellar antigens found for a subpopulation of cells, and the last set of numbers represent flagellar antigens found for another subpopulation. This formula, when given with the genus name - such as Salmonella 30:i:e,n,z₁₅ - identifies the isolate as belonging to a taxonomic subgroup called a serovar. For ease in communication, most serovars are also given informal designations which are written out like species names (but should not be construed as being official species names). For example, the serovar with the preceding antigenic formula is designated Salmonella mjordan (named after basketball star Michael Jordan).

Materials

Heat-killed Salmonella culture belonging to serological group B or C1

Pasteur pipettes

Dropper bottles of "anti-B" and "anti-C1" antisera (antisera prepared against Salmonella cell wall antigens unique to serological groups B and C1)

Petri plates with clear, scratch-free lids

1. On a clean glass slide, draw two circles (about 1 cm in diameter) heavily with a red wax marker.
2. Place the slide in an empty petri dish.
3. With a Pasteur pipette place a drop of the cell suspension within each circle. Make sure the drops appear as even, cloudy suspensions.
4. Without touching the dropper to the cells, place a drop of the anti-B antiserum on one suspension and a drop of anti-C1 on the other.
5. After several minutes, hold up the petri dish and observe the slide from the bottom. Gently tap the dish to effect some mixing of the cell suspension. See figure 14-11. Where there is a reaction between anti-bodies in the antiserum and their homologous antigens on the cell wall of the bacteria, the cells will agglutinate, and the drop will appear to contain many small particles. Where there is no agglutination, the cell suspension will maintain its original, evenly-cloudy appearance.
6. Discard the slide appropriately (or clean it off for reuse) and discard the petri plate into the proper container. (Do not place glass petri plates into the pails with the disposable plastics!)

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14 - 4 Outline of plating and preliminary screening media used in experiment 14

Review of Features Which May be Noted for Many Enteric Differential Media (in Isolation and Differentiation)

Aerobic or anaerobic	Substrate	Microbial activity	Reaction noted	Some Examples
aerobic	various amino acids in peptones	deamination*	alkaline (with pH indicator)	KIA (and probably most media)
	specific amino acid present in large amount	decarboxylation	alkaline (with pH indicator)	XLD Agar, MIO, Lysine Broth
anaerobic	one or more specific sugars	fermentation	acid (with pH indicator)	MacC. Agar, KIA, Lactose Broth, etc.
	thiosulfate	reduction with formation of H2S	black color (with Fe)	Modified MacC. Agar, KIA

^* All enterics (like most common chemoheterotrophs) will deaminate amino acids in peptones, yeast extract, and similar materials. (Deamination of specific amino acids may be tested for as in Phenyl-alanine Agar; see Period 4.

Review of Selective-Differential Media For Initial Isolation

	MacConkey Agar (modified)	Brilliant Green Agar 3	XLD Agar 3
selective agent(s)	bile salts, crystal violet, neutral red	brilliant green	sodium desoxycholate
source of amino acids which may be deaminated 1	peptone, proteose peptone	proteose peptone, yeast extract	yeast extract
amino acid added for detection of decarboxylation 1	none	none	lysine
fermentable sugar(s) 2	lactose (1%)	lactose (1%), sucrose (1%)	lactose (0.75%), sucrose (0.75%), xylose (0.375%)
pH indicator	neutral red: net acid = red, net alkaline = white	phenol red: net acid = yellow net alkaline = red	phenol red: net acid = yellow net alkaline = red
source of H2S	sodium thiosulfate	none	sodium thiosulfate
indicator of H2S production	ferric ammonium citrate	none	ferric ammonium citrate

¹ alkaline reactions.

² acid reactions.

³ optional media; may be used in Exp. 14.

How Acid And Alkaline Reactions Influence The Net pH Observed For Colonies On Various Selective-Differential Media

In the isolation of the enteric pathogens Salmonella and Shigella from food or clinical samples, one usually takes advantage of their gram-negativity (hence the media are selective against gram-positive bacteria) and the fact that they usually react negative for lactose and sucrose fermentation. Therefore, one tends to pick alkaline (non-acid-producing) colonies, ignoring the acid colonies which are produced by coliforms and certain other enterics.

Colonies of usual strains of Citrobacter (a commonly-occurring, non-pathogenic enteric) look identical to those of Salmonella on most isolation media. To assist in differentiating these organisms, XLD Agar is useful.

Shown below is a comparison of Salmonella to selected nonpathogenic enterics, showing the bases for the net pH reactions (and consequently the colony color) one may see on these media, the components of which were given in the preceding section. H₂S production is not pH-related but its detection (with a black color) is a feature of many selective-differential media. (MMAC=Modified MacConkey Agar; BGA=Brilliant Green Agar; XLD=XLD Agar.)

biochemical process	typical Salmonella	typical Citrobacter	typical coliform	process applicable to
Amino Acid Deam.	+ (alk)	+ (alk)	+ (alk)	X	X	X
Lactose Ferm.	-	-	+ (acid)	X	X	X
Sucrose Ferm.	-	-	+ (acid)		X	X
Xylose Ferm.	+ (acid)	+ (acid)	+ (acid)			X
Lysine Decarbox.	+ (alk)	-	+ or -			X
H2S Production	+	+	-	X		X

appearance in	net reaction of typical Salmonella	net reaction of typical Citrobacter	net reaction of typical coliform
MMAC	alkaline(white)+black	alkaline(white)+black	acidic (red or pink)
BGA	alkaline (red)	alkaline (red)	acidic (yellow)
XLD	alkaline (red)+black	acidic (yellow)	acidic (yellow)

Review of Differential Media Used For Preliminary Biochemical Screening

	Kligler Iron Agar (KIA)	Lysine Iron Agar (LIA) 3
source of amino acids which may be deaminated 1	peptone, proteose peptone, beef extract, yeast extract	peptone, yeast extract, lysine
amino acid added for detection of decarboxylation 1	none	lysine
fermentable sugar(s) 2	lactose (1%), glucose (0.1%)	glucose (0.1%)
pH indicator	phenol red: net acid = yellow net alkaline = red	brom-cresol purple: net acid = yellow net alkaline = purple
source from which H2S may be produced	sodium thiosulfate	sodium thiosulfate
indicator of H2S production	ferrous sulfate	ferric ammonium citrate

¹ alkaline reactions.

² acid reactions.

³ optional medium; may be used in Exp. 14.

Explanation Of Reactions Seen in KIA

(First four tubes are demonstrated)

examples	Pseudomonas (a non-enteric)	Morganella Providencia Serratia Shigella	Citrobacter Proteus Salmonella	Escherichia Enterobacter Klebsiella	occasional strains (none for Exp. 14)
glucose fermentation (acid rx.)	-	+	+	+	+
lactose fermentation (acid rx.)	-	-	-	+	+
H2S production (black color of FeS)	-	-	+	-	+
deamination of amino acids (alkaline rx.)	+	+	+	+	+

^* A slight pink or peach color may be seen in the upper part of the slant. Also, much gas (not depicted in this figure) is usually present, indicated by gaps in the butt of the medium.

Key for Enteric Unknowns

	Principal Differentiating Tests				Additional Differentiating Tests
	Phenylalanine Deam.	H2S Production	Lactose Ferm.	Motility	Indole Prod.	Methyl Red	Simmons Citrate	Lysine Decarbox.	Ornithine Decarbox.
Escherichia coli	-	-	+ (w/gas)	+	+	+	-	+	+ or -
Enterobacter cloacae	-	-	+ (w/gas)	+	-	-	+	-	+
Enterobacter aerogenes	-	-	+ (w/gas)	+	-	-	+	+	+
Klebsiella pneumoniae	-	-	+ (w/gas)	-	-	- or ±3	+	+	-
Klebsiella oxytoca	-	-	+ (w/gas)	-	+	- or ±3	+	+	-
Typical Shigella 1	-	-	-	-	+ or -	+	-	-	+ or -
Typical Salmonella 1	-	+	-	+	-	+	+	+	+
Citrobacter freundii	-	+	+2 or -	+	-	+	+	-	+ or -
Proteus vulgaris	+	+	-	+	+	+	-	-	-
Proteus mirabilis	+	+	-	+	-	+	+ or -	-	+
Morganella morganii	+	-	-	+	+	+	-	-	+
Providencia rettgeri	+	-	-	+	+	+	+	-	-

¹ Included on table for comparison only. (Enteric pathogens are not included in your unknown.)

² Lactose fermentation should not be used to differentiate Citrobacter from other organisms. Many

strains of Citrobacter (including the one we generally use) ferment lactose weakly (just a trace of yellow in Lactose Fermentation Broth after 1 or 2 days); such organisms appear lactose-negative on MacConkey Agar and in Kligler Iron Agar.

³ ± = equivocal (orange) reaction in MR-VP Broth.

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14 - 5 Introduction to microbial water analysis

Water, the universal solvent, is essential to life. In drier climes, people will even fight over it (look in a newspaper and read about water rights in Colorado and California). Critical to our modern civilization is the availability of a clean water supply for bathing, drinking and cooking. Unfortunately, many pathogens are transmitted through the water supply. Some of these disease-causing pests enter water from the feces of ill individuals and are then ingested and thereby transmitted to others. Diseases such as polio, typhoid, cholera, hepatitis, shigellosis, salmonellosis and others can spread in this manner. To assure a safe water supply, it is critical to monitor for the presence of these pathogens. However, it would be expensive and time consuming to check the water supply for all of them, instead, an indicator organism is used to assay for fecal contamination. Indicator organisms must have four properties to be useful for water analysis.

1. The only natural environment of the microbe should be in association with feces and it should always be present.
2. It should not grow outside of its natural environment.
3. The bacterium should survive longer than the most viable pathogen, but not so long so that historical events are detected.
4. It should be easy to detect.

Coliforms come closest to fulfilling all these criteria and are the standard indicator organisms used to test for the biological pollution of water. Enterobacter and Klebsiella are able to survive and multiply in the environment and are therefore not the best indicators of fecal pollution. The sole habitat of E. coli and K. pneumoniae, termed fecal coliforms, is the intestines of warm blooded animals. Thus, fecal coliforms are good indicators of fecal pollution and can be differentiated from other coliforms by incubating on selective media at 44.5°C.

Using coliforms, the EPA has developed standards for clean, safe water. These standards vary, depending upon the waters intended use. Drinking water and the water in swimming pools must be of the highest purity. There can be no more than one positive sample (>1 coliform/100 ml) in 40 samples tested in a month and the concentration of fecal coliforms must be zero. But wait, we just said that some coliforms are present in the environment. How can these standards be met? In good quality well water most microorganisms are filtered out as the water percolates from the surface to the well. Unusually high numbers of coliforms in well water may indicate run-off from a polluted area. In the case of surface waters (rivers and lakes), filtration through a sand bed and chlorination remove most microbes. There may also be further steps that need to be taken to insure water safety depending on the treatment plant. Swimming pools, being open surface water, are often contaminated by organisms in the air or by swimming, bacteria-infested humans. Chlorine is added to keep numbers low. Note that the number of permissible coliforms is not zero. This would be difficult to achieve and would provide no additional gain in safety.

Natural bathing beaches and treated sewage are assayed for numbers of fecal coliforms. Total coliform counts are not used as a measure due to the near ubiquitous presence of Enterobacter and Klebsiella in the environment. If the count reaches > 400 fecal coliforms per 100 ml or a monthly geometric average of > 200 per 100 ml, it may indicate a problem in the sewage treatment process or that the beach should be closed. The latter often happens in heavily utilized beaches during the summer.

Testing for coliforms

Presently, several tests are in use to assay for coliforms in water, The oldest of these is the multiple tube fermentation test. In this test three steps are performed; the presumptive, confirmed, and completed tests. A moderately selective lactose broth medium (Lactose Lauryl Tryptose Broth), containing a Durham tube, is first used in the presumptive test to encourage the recovery and growth of potentially stressed coliforms in the sample. If harsher selective conditions are used, a deceptively low count may result. A tube containing both growth and gas is recorded as a positive result. It is possible for non-coliforms (Clostridium or Bacillus) to cause false positives in this medium and therefore all positive tubes are then inoculated into a more selective medium (Brilliant Green Lactose Broth or EC Broth) to begin the confirmed test.

The confirmed test medium effectively eliminates all organisms except true coliforms or fecal coliforms, depending upon the medium and incubation conditions. If a positive result is recorded in these tubes the completed test is begun by first streaking a loopful of the highest dilution tube which gave a positive result onto highly selective Eosin Methylene Blue (EMB) agar. After incubation, subsequent colonies are evaluated for typical coliform reactions.

The multiple tube fermentation test has the great disadvantage of taking 3-5 days to complete. If a municipality has a drinking water crisis, this is too long to wait. This has lead to development of faster, less complex tests. In the membrane filter technique 100 ml or greater of a test sample is passed through a filter with pores small enough to retain all bacteria in the sample. The filter is then placed on a selective medium that allows for the detection of coliforms. The advantages of this technique are the shorter time needed to complete the test (1 day vs. 3 to 4 days), its low cost, the higher accuracy in counting, since the colonies can be enumerated directly from the plate, and its simplicity. Disadvantages are that particulate samples (containing silt or other organic matter) quickly clog the filter, metals and phenols can stick to the filter inhibiting growth, and non-coliforms in the test sample may interfere with the formation of coliform colonies on the plate.

Recently, less complex tests for the detection of coliforms have become available. In the presence-absence test (P-A test), a large water sample (100 ml) is mixed with triple strength LLTB in a single culture bottle. Brom cresol purple is added as a pH indicator. If present, coliforms will ferment the lactose to acid and gas, turning the medium from purple to yellow. To detect coliforms and E. coli, the Colilert defined substrate test can be used. A 100 ml sample of water is mixed with a medium containing ortho-nitrophenyl-β,D-galactoside (ONPG) and 4-methyl umbelliferyl-β,D-glucoronide (MUG) as the only nutrients. If coliforms are present ONPG is metabolized, resulting in a yellow color. If E. coli is present, it will degrade MUG to a fluorescent product that can be detected by observation under long wave length UV-light. Both the P-A test and the Colilert defined substrate test are preliminary and any positive results will warrant further analysis of the offending sample.

The most probable number method of enumeration

In this experiment we will detect coliforms in a water sample using the multiple tube fermentation method. Enumeration of coliforms using this method involves inoculating multiple tubes with a 10-fold dilution series of the water sample and uses the most probable number (MPN) technique to estimate the population. To understand this technique, let us imagine preparing a 10^-1 to 10^-6 dilution series of a culture and inoculating 1 ml portions into tubes containing nutrient broth. After incubation, growth is observed in the tubes inoculated with the 10^-1 to 10^-5 dilutions, but not in the 10^-6 tube. The number of organisms in the original sample is estimated to be less than 10⁶, but greater than 10⁵ bacteria per ml. By inoculating 3 or 5 tubes per dilution and using statistical analysis, a more accurate estimate of the bacterial concentration can be made. This is the basis of the MPN method. Realize that if a 10 ml solution contains 20 organisms, each 1 ml sample will probably not contain exactly 2 organisms. There will be some variation (0, 1, 2, 3 or 4 organisms/ml), but the total of ten 1 ml portions will add up to 20. This is why at lower dilutions, one tube inoculated with a certain dilution blank may show growth (receiving 1 or 2 organisms when inoculated) while other tubes inoculated with the same dilution do not (no organisms in the 1 ml). The number of organisms is assessed by counting the number of positives in the last three dilutions showing growth and then determining the MPN by following the directions on an appropriate table. An excellent explanation of the MPN method has been created by John Lindquist from UW-Madison.

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