Lab One: Staining

Gram Stain
Acid Fast Stain Negative Stain
Gram Stain
Acid Fast
Negative Stain
Positive Stain
Endospore Stain
Capsule Stain
Positive Stain
Endospore Stain
Capsule Stain

 
Safety first:
1.  Never leave flame unattended.
2.  Tie back hair.
3.  Turn off gas after use.
4.  Keep flammable reagents (such as alcohol) away from flame.
5.  Overheating a glass slide will cause it to break.

Procedure for making a slide from a broth culture:

1.  Always hold the slide by the edge to prevent making fingerprints (even when wearing gloves).
2.  Sterilize the inoculating loop by holding it in a flame until it is red.  Let the loop cool near the flame to prevent contamination.
3.  Pass the lip of the test tube in the flame (hold the cap in your hand).
4.  Place a loopful of culture onto the slide.
           Place a loopful of culture onto slide
5.  Pass the lip of the test tube in the flame again and then place the cap on the tube.
6.  Let the slide air dry.
7.  Then heat fix the slide by passing it over the flame four times.  Do NOT heat fix when performing a capsule stain.
8.  Let cool.

Procedure for making a slide from a solid culture

1. Always hold the slide by the edge to prevent making fingerprints (even when wearing gloves).
2.  Sterilize the inoculating loop by holding it in a flame until it is red.  Let the loop cool near the flame to prevent contamination.
3.  Place a loopful of water onto the slide.
4.  Sterilize the inoculating loop by holding it in a flame until it is red.  Let the loop cool near the flame to prevent contamination.
5.  Scrape a small amount of culture and place it in the water on the slide.
         Place a small amount of culture onto slide
6.  Let the slide air dry
7.  Then heat fix the slide by passing it over the flame four times.  Do NOT heat fix when performing a capsule stain.
8.  Let cool.

References:
"Capsule Stain" http://www2.austin.cc.tx.us/microbugz/14capsule.html
"Endospore Stain of Bacillus anthracis" http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/bhttp://www.cat.
cc.md.us/courses/bio141/lecguide/unit1/bacpath/endobant.htmlacpath/endobant.html

"View of Gram Positive and Gram Negative Bacteria"   http://www.arches.uga.edu/%7Esalyee/Slide_of_Gram_Posi tive_and_Gram_Negative_Bacteria.html
"Negative Stain" http://www2.austin.cc.tx.us/microbugz/11negstain.html
"Ziehl-Neelson Staining Procedure" http://www2.provlab.ab.ca/bugs/mycob/afb_stain/znstain.htm