Notes For the 11^th week of Lab April 14 and 16

Bacteriophage Titer (Exercise 25) - You will perform the dilution series below instead of the one described in the manual.

Do not use the vortex mixer after you add the bacteriophage and E.coli to the soft agar since this will cause bubbles in the agar.  Mix the tube by gently shaking it as your TA will demonstrate. This soft agar will be poured over a LB agar plate which provides nutrition for bacterial growth and next week you should see a "lawn" of bacteria with clear areas called plaques which contain the  phage particles. An example of T4 phage plaques in a lawn of E.coli can be seen in the figure below. You will  count the numbers of plaques and determine the number of plaque-forming units PFU/ml in your stock solution. 

                                                               T4 plaques in a bacterial lawn of E.coli

Respiratory Microorganisms (Exercise 23 in Section 14) -  Using a sterile swab you will take a sample from your throat as described in the manual.  You can either swab you own throat or have a lab partner swab your throat.  Tongue depressors are available and try to get as far back toward your tonsils as possible without gagging.  Try not to hit your teeth or other  parts of your mouth with the swab but try to get the whole area of the swab to make contact with your throat.  Roll the swab over the first quadrant of the plate to make sure organisms on the swab will be transferred to the plate.  You will continue with the quadrant  streak technique using a sterile loop for the remainder of the plate.  Next week you will characterize one of the isolated colonies from the plate. Review pages 527 - 529 which describes common groups of streptococci.  Also look at page 403 in your textbook to find the list of normal microflora that you would find in the mouth. You might see gram - cocci like Neisseria, Gram + rods like Corynebacterium, or gram - rods of Fusobacterium.  

Blood agar plates are used for the throat cultures since many  species of Streptococci  (which are part of the normal throat flora) release hemolysins that lyse the blood cells in the plate.  The hemolytic pattern produced by the organisms helps in classifying the different groups of Streptococci.  In lab, we will discuss why an organism would want to lyse blood cells.

If a colony on a plate releases a type of  hemolysin that causes complete lysis of the blood cells and destruction of the cell's hemoglobin in the agar around the colony,  it is called beta hemolysis. You can literally read newsprint through the clear area.  Organisms that show this type of hemolysis are referred to as β-hemolytic streptococci. An example of this type of hemolysis is seen in the following figure.

The organism growing on this plate is called Streptococcus pyogenes  and it causes "Strep Throat".  The area around the colonies is completely clear. There will be a demo plate of this in lab to look at since you can not see the complete clearing in a picture.  One would expect to see very few colonies causing beta hemolysis  from a normal throat culture.  That is why throat cultures are streaked on blood agar from people with a sore throat. If they have an abnormally high level of organisms causing this type of hemolysis, it would be a good indication that the person has "Strep Throat".

Some streptococci release a hemolysin which only partially lyses/damages red blood cells and the hemoglobin in the area on the plate around the colony. This type of hemolysis is called alpha.  An example of this is seen in the figure below.  Organisms showing this type of hemolysis are often referred to as α-hemolytic streptococci.

The organism growing on this plate is called Streptococcus pneumoniae and it causes pneumonia.  As you can see, the area around the colonies is greenish in appearance since the red blood cells have not been completely destroyed around the colony. A plate of this organism will also be in lab for you to examine.  You will probably see many greenish areas around the colonies from your throat culture most likely due to the Viridans Group of streptococci a normal part of the mouth flora.

Many streptococci do not make hemolysins and therefore you do not see any clearing of blood cells around  colonies from these types of  organisms growing on a blood agar plate.  These organisms are sometimes referred to as γ-hemolytic but it is best to call them non hemolytic streptocooci.   An example of an organism that in not hemolytic is seen in the figure below.

The organism on the plate is called Enterococcus faecalis (sometimes seen written as Streptococcus faecalis) and it is a common inhabitant of the gastrointestinal tract.  You will also have a chance to look at this strain growing on a blood agar plate in lab.

ELISA Assay (Exercise 29 in Section 14) -  We will be using a kit  manufactured by Inverness Medical  called "Clearview Strep A Exact ll Dipstick" to determine if a student's throat swab has this organism. Review Exercise 29 which describes the ELISA Technique.  Below is a brief description of the test and directions on how to use it .