Enteric Unknowns Miramar College

Biology 205 Microbiology

Enteric (Greek enteron = intestine) bacteria are comprised of several different genera, but all reside in the digestive tract of mammals. Because the amount of bacteria in the large intestine particularly numbers in the billions, care must be taken to quickly eliminate all but the most obvious pathogens for routine medical care. Typically, enteric pathogens are Gram negative and lack the ability to ferment lactose. There are many commercially available media which are selective for Gram negative organisms and will differentiate them based on lactose fermentation. These media contain a dye that is taken up by lactose fermenters and will pigment these organisms, while lactose non-fermenters remain colorless. Eosin Methylene Blue (EMB) Agar is a medium in which the dyes eosin and methylene blue inhibit Gram positive bacteria. In addition, lactose fermenters will lower the pH of the agar, facilitating their uptake of the dyes, which causes them to display nucleated colonies with dark centers. MacConkey (MAC) Agar contains bile salts and crystal violet dye to inhibit the growth of most Gram positive bacteria and neutral red dye which stains lactose fermenters and provides the pigmentation associated with their identification (Figure 1). The characteristics that distinguish lactose fermenters from lactose non-fermenters are summarized in Table 1. Once the distinguishing characteristic of lactose fermentation has been determined, relatively few biochemical tests are necessary to determine the correct bacterial genus (Figure 2 and Figure 3).

Figure 1: Differential and selective media for the growth of Gram negative enterics, MacConkey (MAC) Agar and Eosin Methylene Blue (EMB) Agar. Media are selective for Gram negatives, by the addition of various dyes and chemicals and differential for lactose fermentation. Typically, lactose fermenters will appear pigmented on these so-called enteric media, and lactose non-fermenters will not. On MacConkey agar, lactose fermenters (left plate, two right side inoculations) appear dark pink. On EMB, lactose fermenters (right plate, two right side inoculations) appear purple or may have a metallic sheen, depending on the organism.

Table 1: Various Selective/Differential media for the isolation and characterization of Gram negative enteric bacteria.

Organism Eosin-Methylene Blue (EMB) agar MacConkey (MAC) agar

Escherichia coli dark center with greenish metallic sheen red or pink Enterobacter sp. similar to E. coli, but colonies are larger red or pink Klebsiella large, mucoid, brownish pink Proteus translucent, colorless transparent, colorless Pseudomonas translucent, colorless to gold transparent, colorless Salmonella translucent, colorless to gold translucent, colorless Shigella translucent, colorless to gold transparent, colorless

Figure 2: Dichotomous key for the identification of lactose non-fermenting enteric bacteria. Genus named listed in white will not be assigned to you as an unknown for this particular laboratory exercise. However, they may be assigned as a Major Unknown organism later in the semester.

Figure 3: Dichotomous key for the identification of lactose fermenting enteric bacteria.

Enterics

For this assignment, you will receive two species in a broth culture. One contains a lactose fermenting Gram negative bacillus and the other contains a Gram negative bacillus which lacks the ability to ferment lactose and produce acid and gas. It will be your job to determine the genus identity of both of these organisms using the biochemical inoculations outlined in the Protocol section of this Lab Exercise and the Dichotomous Key found in Figure 2. You will first isolate these organisms by streaking onto Eosin-Methylene Blue Agar (EMB) and MacConkey agar (MAC). You will be able to identify which is the lactose fermenter. You will then confirm this by inoculating your organisms into separate Phenol Red fermentation broths of lactose and glucose. An alternative method is to inoculate each organism into Russell Double Sugar (RDS) and/or Kligler’s Iron Agar (KIA) slants. Using these multi-test media, you will also be able to determine glucose fermentation and, in the case of KIA, hydrogen sulfide production.

Phenol Red broths, RDS and KIA contain the fermentable sugars glucose and lactose and the pH indicator phenol red. In RDS and KIA the concentration of lactose is 10× the concentration of glucose, and thus glucose is quickly metabolized if the isolate is capable of fermenting it to produce an acidic byproduct and/or gas. If an organism ferments glucose only, the entire tube turns yellow. Because there is a minimal amount of glucose present in the tube, the organism quickly exhausts it and begins oxidizing amino acids for energy. Ammonia is produced and the pH rises. Within 24 hours the phenol red indicator reverts to its original red color on the slant. For this reason, RDS and KIA should not be read prior to 24 hours after incubation. KIA also contains ferrous sulfate (FeSO4), and if hydrogen sulfide (H2S) is being produced, it will react with the ferrous sulfate to form ferric sulfide (FeS2) (Figure 4). Because these tests rely on pH changes in the media, it is important to read the results in a timely manner. As mentioned above, early observations may erroneously lead to the conclusion that an organism is capable of fermenting both lactose and glucose. Further, reading results that are older that 48 hours old may lead to the observation of the phenomenon known as alkaline reversion, whereby the catabolism of amino acids again leads to a raising of the pH which will give false negative results for both glucose and lactose fermentation. Once both glucose and lactose fermentation have been noted, you will test your lactose non-fermenter for motility and urea hydrolysis. Your lactose fermenter will be tested for indole production (using the multi-test medium SIM), urea hydrolysis and citrate utilization. As these media were used as part of your Minor Unknown, you should refer to Minor Unknown Lab for information about these tests and how to interpret them.

Figure 4: Various inoculations onto Kligler's Iron agar slants. Glucose fermentation is noted in the “butt” of the slant- a red butt indicates a lack of fermentation and a yellow butt indicates positive fermentation. Lactose fermentation is noted in the “neck” of the slant, with results similar to that of glucose: red necks are negative for lactose fermentation, yellow necks are positive. Gas production is noted as the slant either splits or is raised up from the bottom of the test tube. Inoculations which are capable of producing H2S form a black precipitate.

Protocol: Enterics

Day One Individual Supplies

Enteric A & Enteric B mixed broth

EMB agar plate

MacConkey Agar plate

1. Streak your cultures on an EMB and MacConkey plate for isolation. Incubate at 37 degrees for 24 to 48 hours.

Day Two

Individual Supplies

incubated EMB and MAC plates

2 Phenol Red lactose broth

1 Phenol Red glucose broth

2 Russell Double Sugar agar or NA slants

1. Gram stain each of your isolates and confirm morphology and arrangement indicative of enteric bacteria.

2. Determine which of the organisms is the lactose non-fermenter. 3. Inoculate both organisms into Phenol Red lactose broths to confirm lactose fermentation. 4. Inoculate the organism you believe is lactose negative into a Phenol Red glucose broth for

glucose fermentation. 5. Inoculate both organisms into NA slants or RDS to confirm lactose non-fermentation and

check for glucose fermentation. 6. Incubate your slants at 37°C for 24 - 48 hours. Day Three

Individual Supplies

incubated NA or RDS slants

Urea broth

SIM

Simmon’s citrate

1. Confirm the lactose non-fermenter, perform the following inoculations: a. Identify your organism’s ability to ferment glucose. b. Inoculate your lactose non-fermenter into urea broth and SIM deep medium. c. Incubate your media at 37°C for 48 hours. Remember that urea broth is a 3–5 day

incubation and should be left in the incubator if a negative result is seen at the end of the 48 incubation period.

2. Confirm the lactose fermenter, perform the following inoculations: a. Inoculate your organism into SIM, Simmon’s citrate and urea broth. b. Incubate all media at 37°C for 48 hours.

Day Four 1. Record all relevant data.

Protocol scheme diagram

Data Collection and Analysis

Enterics 1. Complete the following table for each of your unknown organisms. If you did not perform a

test, indicate that with “n/a.” Biochemical Attribute Organism A Organism B

Gram stain

lactose fermentation

glucose fermentation

motility

urea

H2S production

indole

citrate

1. Identify each of your enteric isolates. My lactose fermenter is ______________________ _____________________. My non-lactose fermenter is ______________________ _____________________. Discussion Questions: Enterics

1. What additives make EMB and MacConkey agar selective and differential?

2. What can be said of lactose non-fermenters on these types of media?

3. What two characteristics separate Salmonella from Shigella? What media can be used for this differentiation?

4. How can acid production by glucose and lactose fermentation be differentiated in the same tube?

5. What is alkaline reversion? Why is it important?

Media Used in Enteric analysis

Enteric Unknowns

medium biochemical/physiological characteristic enzyme reagent(s) positive & negative results

Russell Double Sugar

multi-medium to test for sugar fermentation

fermentative lactose and/or glucose enzymes

[lactose] 10× [glucose] phenol red (in medium),

yellow butt indicates glucose fermentation, yellow neck indicates lactose fermentation,

Eosin-Methylene Blue agar

selective medium inhibits Gram positive; differentiates based on lactose fermentation

various eosin, methylene blue (in medium)

eosin & methylene blue are selective and inhibit Gram positive bacteria, are taken up by lactose fermenters resulting in pigmented and sometimes “nucleated” colonies; lactose non-fermenting organisms remain unpigmented

Kligler iron agar

multi-medium to test for sugar fermentation and H2S production

fermentative lactose and/or glucose enzymes, cysteine desulfurase

[lactose] 10× [glucose] phenol red (in medium), iron salts (in medium)

yellow butt indicates glucose fermentation, yellow neck indicates lactose fermentation, black precipitate indicates H2S production

MacConkey agar

selective medium inhibits Gram positive; differentiates based on lactose fermentation

various bile salts, crystal violet, neutral red (in medium)

bile salts & crystal violet are selective and inhibit Gram positive bacteria, neutral red dye is differential and pigments lactose fermenting organisms red; lactose non-fermenters are non pigmented

Phenol Red Glucose Broth

Differential media to distinguish glucose fermentation and acid and gas production

various Phenol Red, glucose Acid production turns broth yellow, gas production is visible as a bubble in inverted Durham tube

Phenol Red Lactose Broth

Differential media to distinguish lactose fermentation and acid and gas production

Beta galactosidase Phenol Red, lactose Acid production turns broth yellow, gas production is visible as a bubble in inverted Durham tube

SIM H2S Production; indole production; motility

cysteine desulfurase; trytophanse

iron salts (in medium); Kovac’s reagent

black precipitate indicates H2S production; red color change with Kovac’s reagent indicates indole production from tryptophan; cloudy throughout indicates motility

Simmon citrate agar

citrate as a sole source of carbon citrase bromthymol blue (in

medium) blue color change indicates presence of citrase

urea broth production of urease urease phenol red color change to hot pink/cerise color indicates presence of urease