Second Lab - Experimental Protocol

2.1. Special Materials:

2 BA plates, demonstration plates of α&β hemolytic bacteria

2.2. Procedure:

1. Read the BA plates from the first lab. Observe and record the colony morphology and characteristics of the various organisms present. Record any types of hemolysis (see the demonstrations) you observe on LAB RESULTS page at end of this notebook. Try to determine the number of different organisms present on the basis of colony characteristics, and record the frequency of each type and study it according to the key provided at the end of the manual. You should be able to observe at least 4 to 5 different colony morphologies. Confer with an instructor if you don't.
2. Obtain pure cultures of 4 different colony types. Accomplish this by touching a colony with the tip of a sterile loop to obtain an inoculum for streaking to single colonies. Streak to single colonies on 1/2 of a new, labeled BA plate. If you can't obtain 4 different types of colonies from your plate, borrow from a neighbor.
   
   To help you with your observations of growth on the BA plates, below is a listing of colony characteristics to note.
   1. Color: white, creamy, yellow, red, etc.
      -pigment diffusible or confined to colony.
   2. Opacity: opaque or translucent.
   3. Size: pinpoint, 1 mm, 5 mm, etc
   4. Colonial morphology:
      1. Edge of colony: smooth or rough?
      2. Surface of colony: mucoid (wet), smooth, rough, domed, flat, umbilicate (depression in center)?
   5. Hemolysis (on blood agar): complete, (β, yellowish, agar color); partial (α, green); or none (γ). α ƒnƒnhemolysis is a greening of the agar indicating partial hemolysis of the blood. β hemolysis is a total clearing or use of the heme in the blood. The agar is yellow and clear. Streptococci are classified as α or β depending on the type of hemolysis. γ hemolysis means there has been no hemolysis at all.
   6. Odor: note any odor. Some bacteria have a very distinctive odor. Pseudomonas aeurginosa has a "grape-like" fruity odor. Many think that Proteus mirabilis smells like chocolate cake. A blood culture that has a sulphur-like smell may well have Salmonella typhi. Many anaerobes, bacteria that grow without oxygen, have very distinctive odors as well.

The following is a guide to help you in identifying the organisms you have isolated from the BA plates. The same information is presented as a flow chart at the end of the manual.

1. Colonies about 1 mm in diameter or larger
   1. Gram+ cocci
      1. Colonies creamy and opaque, golden or white, rarely yellow. Cocci in clusters.
         1. Coagulase test+, colonies usually golden, sometimes β-hemolytic. Staphylococcus aureus
         2. Coagulase test-, colonies white, rarely yellow. Staphylococcus epidermidis
      2. 2) Gram- cocci
         1. Colonies of various pigmentation. Neisseria species. Typically diplococci.
      3. Gram+ rods
         1. Colonies rough, irregular, non-hemolytic. Rods pleomorphic. Corynebacterium species
         2. Colonies very large, irregular, often hemolytic. Rods large, often sporulating in old cultures. Bacilli
      4. Gram- rods
         1. This category is not usually found in throat swabs grown on BA plates. Consult your instructor if you think you have isolated an organism from your throat that forms large colonies on BA plates and is a Gram- rod.
   2. Colonies pinpoint in size
      1. β-hemolytic
         1. Colonies opaque and hard, white. Gram+ cocci in chains. Streptococcus pyogenes
         2. b) Colonies translucent and soft. Gram- rods. Haemophilus hemolyticus
      2. α-hemolytic (Gram+ cocci in chains or pairs)
         1. No growth around optochin disc. Streptococcus pneumoniae
         2. Growth around optochin disc. Streptococcus viridans
      3. Non-hemolytic
         1. Gram+ cocci in pairs or chains - "non-hemolytic Streptococcus"
         2. Gram- rods. Haemophilus species

NOTE: To make the best use of this exercise, you should relate your lab work to your text book reading.

3.1. Special Materials:

oxidase reagent, plates with Neisseria

3.2. Procedure:

1. Carry out the oxidase test on colonies on your chocolate agar plate. Apply a 1% solution of the oxidase reagent directly onto a few isolated colonies. Observe colonies for 6-10 minutes for evidence of color change. Colonies that become pink, then purple, and finally black, are considered "oxidase positive." Record Results.

   The Oxidase Test
   Purpose:	Aid in identification of Neisseria and Pseudomonas aeruginosa
   Principle:	Detection of the presence/absence of indophenol (cytochrome) oxidase
   Mechanism:	Indophenol oxidase oxidizes reagent to purple color
   Reagent:	1% tetramethyl-p-phenyldiamine
   Method:	Add a drop or two of reagent to colonies on agar plates
   Interpretation:	Positive - colonies turn pink, then purple, and finally black Negative - no or very slight color change

2. Pick from the positive colonies and prepare a smear for Gram straining. Record results

4.1. Special Materials:

1 mannitol salts agar plate, sterile swab

4.2. Procedure:

Using a sterile swab, culture your nasal flora onto a mannitol-salts agar plate. This is a selective plate: the high salinity inhibits the growth of most normal flora, especially Streptococcus sp. It is also a differential medium, allowing you to distinguish Staphylococcus epidermidis (mannitol-) from Staphylococcus aureus (mannitol+; plate changes color from orange-pink to yellow), a potential pathogen.