WEEK 3 LAB: Guide Questions

Identify the steps taken in the preparation of a bacterial smear to prevent

contamination of the culture and the preparer of the smear.

Procedure:

 Begin this exercise by disinfecting the laboratory desk. Then place a clean
sheet of paper towel or absorbent material over the area to be used.
Should a spill occur, the paper will absorb the liquid and can be easily
disposed.
 Get a new, clean, and dry glass slide. Using a wax marking pencil or felt
permanent marker, mark off the areas of the slide where smears will be
placed.
 Obtain cultures of the bacteria, and place the tubes in a suitable rack on
the bench. Sterilize your inoculating loop. If agar slant cultures are used,
place a loopful of water into one area of the slide. If broth cultures are
used, no additional liquid is necessary. Light the Bunsen burner.
 Firmly hold the loop as you would hold a pencil, and place it nearly
vertically into the blue tip portion of a Bunsen burner flame for a few
seconds or until it glows red hot. Allow the loop to cool several seconds.
 Take the bacterial culture in the opposite hand, remove the cap. Place the
tip of the tube in the flame for a few seconds. This will burn off any dust or
lint and kill any airborne organisms that might happen to fall into the tube.
 Insert the lop into the tube, and touch it every gently to the main portion of
the slant. Be careful not to dig into the medium or scrape it. If a broth
medium is used, dip the loop carefully into the liquid. Remove the loop, re-
flame the tip of the tube briefly, then replace the cap.
 Mix the loopful of bacteria with the loopful of water on the slide, and swirl
the liquid out to the area of a five-centavo coin. For broth cultures, simply
swirl the loopful of bacteria on the marked slide area. Complete the
procedure by reflaming the loop. You now have a bacterial smear.
 Prepare other smear in the remaining spaces with different bacteria.
 Air-dry the slide until all the liquid evaporates.
 Heat-fix the smears by quickly passing the slide through the Bunsen
burner flame three times. The heat should contact the underside of the
slide. This procedure kills the bacteria that may still be alive, facilitates
stain penetration, and fixes cells to the slide so they do not wash off when
stained. The slide is now ready to be stained.
1. Summarize the fundamental theory of simple staining.

 Bacterial smear is process where it is stained with reagent to produces a

distinctive contrast between the organism and the background.

2. Judge the result of placing a large drop of water on the slide instead of a loopful.

 Placing large amount of water on the slide can make you to wait longer to
evaporate the water and it has a tendency to overflow the water from the
slide.

3. Discuss the possible outcomes if an air-dried smear were not heat-fixed before
proceeding with the staining step.

 If an air–dried smear were not heat-fixed before proceeding to the

staining step, it cannot kill any bacteria that may still be alive, Facilitates
statin penetration and fixed cells to the slide so they do not wash off when
stained.

4. When you observe your stained smear at 1000X, you see two different shaped
bacteria; some are rod shaped while others are spherical. Explain this result
considering the original source culture was pure.

 Gram-positive and Gram-negative bacteria stain differently because of

fundamental differences in the structure of their cell walls. The bacterial
cell wall serves to give the organism its size and shape as well as to
prevent osmotic lysis.