-Many different stain procedures
Simple/Complex
General/Specific
General steps: Smear, Air Dry, Fix, Stain
Be sure to use Aseptic Technique at all times.
Smear:Play Demo
Place thin film of bacteria on slide.
If broth, smear directly on slide
If agar, suspend in drop water then smear on slide
Tips:
-Put at one end of the slide.
-Put on thin film.
-If beads rather than spreading smoothly, then slide may be dirty.
Air Dry:
Heat Fix: Play Demo.
Pass thru the flame quickly.
Tips:
-Does not take much to heat fix.
-Too long and the bacteria “burn” and/or stain irregularly.
Stain:Play Demo.
Tips:
-Put one end of the slide on the lip of the staining tray.
-Let stream from wash bottle flow down over the bacteria.
-Wash both front and back of slide.
-Blot dry using bibulous pad. Do not wipe dry, as may wipe off bacteria.
Viewing:
Tips:
-Use oil at 100x to view bacteria
-Do not use coverslip. Put drop of oil directly on bacteria.
-Make sure only 100x lens is used with the oil.
-Once the oil is on, it is difficult to reuse that slide. Therefore, if you cannot focus at 40x do not go to 100x.
-Trouble focusing at 40 usually means there is oil on the lens.
-Gram positive is purple. Gram Negative is pink
Ideally.
Bacillus +, bacillus
Staphylococcus +, coccus
Neisseria -, diplococcus
Escherichia -, bacillus
Remember the various reasons as to why get “false” results.
Questions/problems
1. a. How stable is the gram stain characteristic of a bacteria?
b. What factors could influence the outcome of the
gram stain?
2. How would you explain a gram positive appearing gram negative? Would
your Bacillus stain differently at the after growth for several days.
3. How would you explain a gram negative appearing gram positive?
4. How would you explain no bacteria appearing on the slide after staining?