BIO 304                  Lab Worksheet 2                Spring 2006

 

NAME________________________________________________

 

Coverage:

Ex. 10: Aseptic Technique

Ex. 3 Smear Preparation

Ex 5 Gram Staining

 

Using the lab manual and/or your notes, answer the questions as completely as possible.

 

Aseptic Technique:

 

What is the primary function of a:

 

broth? supports the growth of large numbers in a small tube

 

 

 

stab? (aka semi-solid agar, or deep)

          to test for oxygen requirements or motility

 

 

 

 

slant?

          storage

 

 

 

 

What tool does one use to inoculate a:

 

stab:            needle                    slant:            loop                       broth:  loop

 

 

 

Smear Preparation:

 

Complete the following sentences:

 

To make a proper smear from a liquid culture, transfer a very____small____amount of culture to a clean glass________slide__________and spread it out.  Allow the smear to _______air_______dry COMPLETELY.  After the smear is dry, _____heat fix________it, then continue with the desired staining procedure.

 

Why is a smear allowed to air dry?  To preserve the shape of the sample, as close as possible to the true shape

 

 

What is the purpose of heat fixing?

          to kill the cells, and stick them to the slide

 

 

Bacteria can be seen without staining.  Why then, was Koch’s recommendation for fixing and staining important for the study of microbiology?                  To provide more contrast for easier viewing, and to promote consistency between investigators

 

Gram Staining:

 

See your lab manual, page 40, Question 4.  Please recreate the table there, and complete the steps and appearance.

 

Step # and name

Chemical

Gram Positive

Gram Negative

1 Primary stain

Crystal violet

Blue

Blue

2 Mordant

Gram’s iodine

Blue

Blue

3Decolorization

Alcohol

Blue

Clear

4secondary stain

Safranin

Blue

Red

 

 

Draw the structure of the Gram Postive and Gram Negative bacterial cell envelope below.  What are the two reasons that the CV-I complex is washed out of Gram negative cells?

 

Postive, thick peptidoglycan

Negative, thin peptidoglycan, outer membrane

 

the decolorization step strips the outer membrane and the CV-I complex is not retained in the cell due to the thin peptidoglycan layer

 

 

 

 

 

 

Which step can you leave out, and still determine the result of the Gram reaction?

 

Secondary stain

 

 

See page 40, lab manual.  Answer Critical Thinking questions 1 and 2 here:

 

1.         Identically shaped adjacent cells that stain differently in the Gram stain occur because at the time of staining, some of the cells in the pure culture were dead.  Dead cells do not stain appropriately, because their peptidoglycan layer has degraded.

 

 

 

 

2.       You are observing a mixed culture of Gram negative rods and Gram positive cocci