Question

You are given a mixed culture of two organisms in a small tube of TSB. These two organisms include a Gram-negative strain (your bacterial strain to identify) and a Gram-positive strain (a contaminant). Your first task is to isolate these organisms which will help you identify which Gram-negative strain you are working with. 

1) You inoculate two streak plates on TSA media and MacConkey Agar using aseptic technique. For each medium, state whether the medium is selective, differential, or neither and explain if this medium will help you isolate your Gram-negative organism as a pure culture.  

TSA - 

MacConkey Agar-

2) Your inoculated TSA and MacConkey plates were incubated overnight at 37°C. For each of the plates, describe the colony margins of the colonies seen in the photographs below using the terms on page 22 of the lab manual. Note: There may be more than one type of colony per plate. 

TSA Colony Morphology - 

MacConkey Colony Morphology -

You select an isolated colony of each organism from your TSA plate and perform a Gram stain on a glass slide which you view under the microscope using the 100X objective lens. You find two distinct types of colonies. 

Based on the photographs above describe the shape (cocci/bacilli/etc) and size (length and width using the micrometer in the photo) of your two microorganisms and state whether they are Gram positive or Gram negative. 

Once you have figured out which of your two organisms is the Gram-positive contaminant and which is the Gram-negative bacterial strain, you prepare a streak plate of your Gram-negative bacterial strain on EMB Agar (Eosin Methylene Blue). Below state whether EMB Agar is selective, differential, both, or neither and explain how this will help you to isolate and identify your Gram-negative organism. 

EMB Agar -

3) Your EMB Agar plate was inoculated overnight at 37°C. You now have a pure culture of your Gram-negative bacterial strain to use for inoculations of several biochemical tests. You should be familiar with these biochemical tests from our previous experiments throughout the semester. Based on what you know about EMB Agar and the photograph below of your incubated EMB plate are there any Gram-negative organisms you can eliminate from the possibilities for your unknown bacterial strain? 

Now that you have a pure culture of your Gram-negative organism you perform an assay of biochemical tests and inoculate several different types of media. For each bacterial medium below describe what is being tested for, what a positive result looks like, and any reagents which must be used to interpret the test. A few sentences should be sufficient for each medium. 

Phenol Red Glucose, Sucrose, and Lactose Broths - 

For Phenol Red Broths: Specifically describe what is being tested, what the medium's color indicates, and what the small inverted glass vial (Durham tube) is used for. Also describe what a positive result looks like and what (if any) reagents are used to interpret the results. Please write in the space below. 

SIM Media - 

For SIM Media: Specifically describe what three things are being tested for, what a positive result looks like for each of them, and what (if any) reagents are used to interpret the results for each of them. Please write in the space below.

MR-VP Broth-

 For MR-VP Broth: Specifically describe what two things are being tested for, what a positive result looks like for each of them, and what (if any) reagents are used to interpret the results for each of them Please write in the space below.

Simmon's Citrate Agar - 

For Simmon's Citrate Agar: Specifically describe what is being tested for, what a positive result looks like, and what (if any) reagents are used to interpret the results. Please write in the space below.

4) You return to lab the next day and your biochemical tests have all been inoculated overnight at 37°C. Based on the photos below and your knowledge of each biochemical test, fill in the table with what is being tested for and state whether the results are positive or negative for your organism. 

Based on your results so far you may already be able to narrow down which organism you have. However, others will need to perform additional biochemical tests to further determine which organism they have isolated.

In the space below circle which of the following tests (if any) you would perform next. Describe what is being tested for, what a positive result would look like, and what reagents (if any) would be used to interpret the results. You can use the following page if you need more room. 

Once you have finished interpreting the results contained within this exercise please fill in the name of the microorganism you believe you have isolated. If you chose to perform an additional test on the previous page write the name of the organism(s) which would have resulted from a positive and negative outcome for that test. 

Name of Microbe(s):

Answer 1

Exercise 13

1.

Isolation of bacteria is accomplished by growing ("culturing") them on the surface of solid nutrient media. Such a medium normally consists of a mixture of protein digests (peptone, tryptone) and inorganic salts, hardened by the addition of 1.5% agar. Examples of standard general purpose media that will support the growth of a wide variety of bacteria include nutrient agar, tryptic soy agar, and brain heart infusion agar. A medium may be enriched, by the addition of blood or serum. Examples of enriched media include sheep blood agar and chocolate (heated blood) agar.

Selective media contain ingredients that inhibit the growth of some organisms but allow others to grow. For example, mannitol salt agar contains a high concentration of sodium chloride that inhibits the growth of most organisms but permits staphylococci to grow.

Differential media contain compounds that allow groups of microorganisms to be visually distinguished by the appearance of the colony or the surrounding media, usually on the basis of some biochemical difference between the two groups. Blood agar is one type of differential medium, allowing bacteria to be distinguished by the type of hemolysis produced. Some differential media are also selective, for example, standard enteric agars such as MacConkey and EMB agars, which are selective for gram-negative coliforms and can differentiate lactose-fermenting and non-lactose-fermenting bacteria.

Trypticase soy agar or tryptone soya agar (TSA) and Trypticase soy broth or tryptone soya broth (TSB) with agar are growth media for the culturing of bacteria. They are general-purpose, nonselective media providing enough nutrients to allow for a wide variety of microorganisms to grow. They are used for a wide range of applications, including culture storage, enumeration of cells (counting), isolation of pure cultures, or simply general culture.

MacConkey agar is an indicator, a selective and differential culture medium for bacteria designed to selectively isolate Gram-negative and enteric (normally found in the intestinal tract) bacilli and differentiate them based on lactose fermentation. The crystal violet and bile salts inhibit the growth of Gram-positive organisms which allows for the selection and isolation of gram-negative bacteria. Enteric bacteria that have the ability to ferment lactose can be detected using the carbohydrate lactose, and the pH indicator neutral red.

2.

a) TSA agar colonies morphology:

Size- punctiform, Form- circular, Margin- entire, colour- white, opacity- translucent colonies

MacConKey agar colonies morphology:

Circular, raised, entire, cream colour, translucent colonies with pink colour surrounding.

b). Organism 1-

Shape- bacillus, Size- smaller (1-2 micrometer), Gram negative

Organism 2-

Shape- spiral long, Size- lager than organism 1 (4-5 micrometer), Gram positive

c).

EMB agar:

Eosin methylene blue (EMB, also known as "Levine's formulation") is a selective stain for Gram-negative bacteria. EMB contains dyes that are toxic to Gram-positive bacteria. EMB is the selective and differential medium for coliforms. It is a blend of two stains, eosin and methylene blue in the ratio of 6:1. EMB is a differential microbiological medium, which slightly inhibits the growth of Gram-positive bacteria and provides a color indicator distinguishing between organisms that ferment lactose (e.g., E. coli) and those that do not (e.g., Salmonella, Shigella). Organisms that ferment lactose display "nucleated colonies"—colonies with dark centers.

3.

The colonies in the EMB agar plate looks like nucleated colonies, i.e colonies with dark center.