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LABORATORY 6

Objectives: Upon successful completion of this laboratory, you will be able to:

1. Describe the cultural characteristics that is distinctive for selected species of the following gram-negative genera: Proteus, Serratia, and Pseudomonas.
2. Understand how to inoculate a variety of media for gram-negative identification.
3. Understand the initial process required for the identification of gram-negative enteric organisms.

1. Observe the demonstrations and plates showing swarming by Proteus sp. and pigment production by Pseudomonas aeruginosa and Serratia marcescens.
2. Observe Biochemical Reactions via the Classical Tube Methods

• MacConkey Agar Plate—Mac
• Triple Sugar Iron Agar Slant—TSI
• Indole/Methyl Red/Voges-Proskauer/Citrate—IMViC tests
• Urea agar
• Motility Medium, Motility/Ornithine/Indole Medium (MOI), or other.
• Oxidase reaction

3. Perform a Gram stain on Unknown No. 2.

4. Inoculate tube media for the identification of Unknown No. 2.

5. Streak Unknown No. 3 (it contains two organisms) on one MacConkey, one CNA, and one BAP plate.

The gastrointestinal tract of humans is the normal habitat for many different Gram-negative bacilli (rods). Most of these belong to the family Enterobacteriaceae, are facultative anaerobes, and ferment glucose. The many species of this family exhibit a wide variety of biochemical reactions. The near-continuum of these traits reflects the considerable exchange of genetic material among the species during their evolution.

Biochemical similarities form the basis of the classification shown below. In this lab exercise you will examine some of the biochemical reactions clinical bacteriology laboratories use to identify those organisms marked below with an asterisk (*).

Several other genera of Gram-negative bacilli that are found in the GI tract differ from the Enterobacteriaceae by their strict requirement for oxygen or by their metabolism of carbohydrates. Some of these other organisms include:

The genera Campylobacter and Helicobacter contain microaerophilic organisms (require reduced O₂ levels) that often require CO₂ and elevated (42^oC) temperature for growth; therefore they are easily missed during routine culture.

Finally, not included in the list above but equally prominent in the normal gastrointestinal tract, are staphylococci, streptococci, and most importantly, anaerobes. The anaerobic species make up the bulk of the stool microflora flora and outnumber other species by several orders of magnitude. Anaerobic organisms responsible for infection will be covered in the lecture portion of this course.

A number of the Gram-negative bacilli in the above lists are not normally pathogenic within the gastrointestinal tract but do produce disease opportunistically when they (1) invade the urinary tract or blood stream, (2) enter the peritoneum through a break in the intestinal wall, (3) become trapped behind an intestinal obstruction, or (4) ascend the biliary tract. In debilitated or immunologically defective patients these organisms may invade other parts of the body and produce serious infections.

These demonstrations will acquaint you with the appearances of the various reactions of bacteria in media and tests that are used for identification of gram negative bacteria in the absence of commercial systems. In order to be able to identify Unknown #2, you will need to understand how to inoculate these media and how to identify and interpret the various biochemical reactions produced. Descriptions of these media re also found in Appendix 2.  

1. MacConkey (Mac) agar plate has isolated colonies from bacteria that can and cannot ferment lactose. Mac agar is a selective and differential plating medium recommended for use in the detection and isolation of Salmonella, Shigella and other enterics from stool specimens, urines and other materials. This medium contains peptone, lactose, bile salts, neutral red, and crystal violet. Gram-negative bacteria are differentiated by their ability to ferment lactose. Observe for isolated colonies: lactose fermenters which appear bright pink and may be surrounded by a zone of precipitated bile (due to the action of acids, produced by fermentation of lactose, upon the bile salts and subsequent absorption of the indicator neutral red). Non-lactose fermenters utilize the peptone, thus yielding an alkaline reaction which changes the color of the medium to clear or yellow. These colonies are uncolored and transparent. Gram-positive organisms are inhibited by inclusion of the dye crystal violet and bile salts in the medium. The Diagnostic Microbiology Laboratory uses MacConkey plates for primary isolation of organisms from specimens with suspected Gram-negative infections.
2. Triple Sugar Iron (TSI) tubes were inoculated from an isolated colony or a pure broth culture by streaking the surface of the slant. After streaking the slant, and without removing loop from the tube, the agar butt was stabbed with the loop by starting at the base of the slant and stabbing to the bottom of the tube. These tubes were incubated for 24 hours at 37^oC and then refrigerated until this laboratory period. Observe the tubes for the various reactions that may be demonstrated in this medium (refer to the accompanying table). Reactions on the TSI medium must be read after 24 hours of incubation at 37^oC. Prolonged incubation may result in reversion of an acid reaction on the slant to alkaline.

Reactions in TSI Agar

Reaction	Interpretation
–Alkaline slant (red), acid butt (yellow) (K/A)	Glucose fermented
–Acid slant and butt (medium yellow throughout) (A/A)	Lactose or sucrose, or both fermented
– Alkaline slant and butt (medium entirely red) (K/K)	None of the three sugars fermented
– Gas bubbles in butt, medium sometimes split (G)	Gas (CO2) produced as a by-product of fermentation
–Blackening of the butt and/or stab (H2S)	Hydrogen sulfide produced

 

 

3. IMViC is a mnemonic coined to designate the combined results of four different tests: Indole, Methyl Red, Voges-Proskauer, and Citrate. The "i" was inserted for easier pronunciation. (Read Appendix 2: Culture Media and Biochemical Tests for a separate discussion of each test.) The method for performing each biochemical test is discussed under the types of medium used for the test. One tube of each of three types of media was inoculated and incubated at 37^oC for the indicated times. Note that only one tube of MR-VP broth is needed. For Indole, MR + VP tests, cultures must be incubated for at least 48 hours. Your cultures were incubated for 72 hours.
4. Indole Production: Observe the 48-72-hour MOI media to which 6-8 drops of Kovac's Reagent have been added. Note the differences between a positive and a negative reaction. Indole reacts with the reagent to give a red (positive) color in the layer above the media.
5. Methyl Red Test: Observe the 48-72-hour MR-VP broth cultures (minus one ml of culture used for the VP test) to which 6-8 drops of methyl red indicator were added. A reddish color indicates a pH of less than 4.5 (positive result).
6. Voges-Proskauer Test: The VP test is performed on one ml of a 48-72-hour MR-VP broth culture. After addition of 15 drops of a-naphthol (Reagent A) and 10 drops of Reagent B, a positive reaction is evidenced by development of a red color in the top layer of the medium which gradually spreads throughout the tube. A negative reaction for acetylmethylcarbinol production shows no evidence of color after 30 minutes in the presence of the reagent
7. Citrate Test: Observe the Simmons citrate agar slants for positive and negative reactions indicating utilization of citrate as the sole carbon source. These tubes can be read after 24 hours of incubation. The blue color (positive reaction) is indicative of an alkaline change in pH as the result of cleavage of citrate. To inoculate this medium, it is only necessary to streak the slant. It is not necessary to stab the butt as with the TSI medium.
8. Urea agar slants: Observe the urea agar slants and note the positive reaction (red color) indicating the production of the enzyme urease. This medium is used for identification of Proteus species and can be read after 24 hours of incubation. It can also be used to differentiate species of Yersinia. Streak the slant with an isolated colony from your EMB or Mac plate.
9. Motility medium. In this semi-solid agar, motile organisms spread throughout the agar, while non-motile organisms grow only along the path of the inoculating needle. Depending on the type of motility medium use, there may or may not be dyes to aid in visualizing the growth. With a straight inoculating wire (not a loop) inoculate the motility medium from colony or a liquid culture by stabbing the semi-solid agar to about 1/2 inch below the surface. Incubate at 37^oC for 24 hours and observe for motility. If the test is negative, leave the tube at room temperature for another day. Some organisms synthesize their flagellar proteins optimally not at 37^oC but at room temperature.
10. Oxidase reaction. This tests for bacterial cytochrome oxidase. Open the vial of Oxidase Reagent. Place a drop or two of the reagent on a colony and observe for color change. Oxidase activity can also be visualized by transferring a portion of a colony to a piece of filter paper and performing the test on the bacteria on the filter paper.

A culture of an unknown Gram-negative bacillus will be assigned to each student.

C. Unknown No. 3

One mixed culture containing two unknowns (Gram-positive organisms, Gram-negative organisms, or a mixture of both types) will be assigned to each student.

1. Characteristics on Mac agar:

 

2. Gram stain (circle one) : gram- positive, gram- negative, gram-variable
   Describe morphology

 

3. Media inoculated for identification and biochemical reactions:

 





 

 

 

 

 

 

4. List possible organisms from above test results
   
   
   

 

5. Further tests done to complete identification (record results)
   
   

 

 

6. Definitive identification of organism

/Total Points (0.5)

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