Bacteriology 102:

Media
Review
Questions

1.  Relevant to formulation of selective-differential isolation media and Experiment 14:  You wish to exploit certain properties of the difficult-to-isolate bacterium Excalibacterium (an enteric) in order to help you detect and isolate it from samples which are highly contaminated with other enterics. You decide to start with MacConkey Agar which you know contains lactose as the only fermentable sugar. Peptone is another medium ingredient which you recall; it contains a mixture of various amino acids – none in any especially high amount. Following is a table showing important genera to consider in this situation:

• Why would you expect Edwardsiella, Aquamonas and Excalibacterium to produce an alkaline reaction on MacConkey Agar?
  
• As these 3 genera don't ferment or respire lactose, how can they grow on MacConkey Agar?
  
• What would be the best choice for a sugar to add to MacConkey Agar which will assist greatly in the differentiation of Excalibacterium colonies from all of the other organisms on the table?
  
• If lysine were to be included in the medium in a relatively large amount, what effect would this have on the pH reaction associated with Excalibacterium colonies? What about arginine?
  

2.  Another question relevant to differential media and Experiment 14:  You are in an enteric lab out in the real world, and you are picking colonies off plates of selective-differential isolation media for further testing. These plates had been inoculated with environmental samples, and we expect a variety of enterics to be present. We also expect some colonies of that pesky Pseudomonas to be present also.

Now, the organism you are specifically after is Sorgobacter, an enteric with one or more characteristics that allow it to be differentiated from all other enterics – as seen by the reactions in the following table:

As part of the enteric isolation routine, you plan on picking colonies into a screening medium, such as Kligler Iron Agar (KIA) before doing a lot of specific tests. Before you do that, you decide you have time to make a modification of KIA that will allow you to decide whether or not you have Sorgobacter, just from the appearance of the modified KIA after incubation.

• You know that KIA already has two sugars in it – lactose and glucose – and the glucose is present in a relatively small amount. Which one of the following sugars would you add to the ingredients in KIA (in a relatively large amount, like lactose) such that Sorgobacter will look different from all of the other enterics and also Pseudomonas? Fructose, galactose or mannitol?
  
• Briefly describe the appearance of Sorgobacter growing in this modified KIA. That is, would you expect an acid or alkaline reaction in the aerobic (slant) region? And what about the anaerobic region (i.e., the "butt")?
  
• What would be the pH reaction in the aerobic (slant) region for all of the other enterics? Acid or alkaline?
  
• If (for whatever reason) you were to add one of the amino acids to the medium, which one could allow Sorgobacter to show an alkaline reaction in the anaerobic (butt) region? Arginine or lysine?

3.  Here is an easier KIA-related question:  You have a Kligler Iron Agar (KIA) slant which you inoculate with an enteric that ferments glucose (the sugar that is in the relatively low concentration in KIA) but not lactose (the sugar in the high concentration). The organism also deaminates some of the amino acids in peptone (as do all enterics), and it also decarboxylates lysine but does not produce H₂S.

• You accidentally pour about 10 ml of sterile mineral oil over the medium, completely immersing the slant. What would be the expected appearance of the medium (in the slant and in the butt) after incubation at 37°C for 1 day? (For "appearance" you can indicate colors or the expected pH – acid, alkaline or neutral.)
  
• What would one normally see if the mineral oil were kept off the medium?
  
• If you were to make up some KIA slants which contain a large amount of added lysine, and then inoculate a tube with the same organism (keeping the mineral oil off the tube!), what would be the expected appearance after 1 day at 37°C?

4.  Here is yet another KIA-related question:  You isolated an enteric from beautiful Lake Splammo, and your Kligler Iron Agar culture of the isolate has a red (alkaline) slant and a yellow (acidic) butt after one day of incubation at 37°C. To begin the identification process, you consider the reactions on the following table:

• From the results of just the pH-related reactions, you know that the isolate would not be identified as              or             , and you can also see that the isolate is positive / negative (circle one) for H₂S production.
  
  
• Your lab partner has been experimenting with differential media and has added a somewhat large amount of mannitol and ornithine to her KIA formulation, and she also got the same result with the same isolate as you got in your KIA tube with the normal formulation. According to the table, the organism would be identified as this genus:             .
  

5.  Relevant to formulation of selective isolation media and our enrichment-isolation experiments:  You have been commissioned to isolate the following type of bacterium from soil:

• Would you want to heat-shock the soil sample?
  
  
• Which two of the following would you include in your isolation medium (i.e., the plating medium on which you obtain isolated colonies from having inoculated it with your sample) in order to recover the organism with minimum interference from other organisms in the soil?
  
  • a nitrate compound
    
  • glucose
    
  • starch
    
  • peptone
    
    
• If you incubated your plates aerobically, what would you expect to find among the colonies on your plates? Choose one or more:
  
  • the type of organism whose requirements are listed above
    
  • those that break down starch, aerobically respire the products of starch hydrolysis, and use nitrate as the N source
    
  • those that break down starch, ferment the products of starch hydrolysis, and use nitrate as the N source
    
    
• If you incubated your plates anaerobically, what would you expect to find among the colonies on your plates? Choose one or both:
  
  • the type of organism whose requirements are listed above
    
  • those that break down starch, ferment the products of starch hydrolysis, and use nitrate as the N source
    
    
• Discuss how you would test your isolates (the cultures you are deriving from your isolated colonies) to see if they meet the initial requirements. Considering the conditions in which you would have incubated your plates, are any of these requirements met already? In testing for oxygen relationships (to see if your isolates are strict aerobes or not), would you want to include nitrate in your medium?