General Microbiology Lab Briefing

Exercise 9:  Staining Bacteria

We use three staining techniques in General Microbiology:

1. Simple Stain — uses one stain and all bacteria are the color of the stain.  Is not a differential stain.
   • Preparation of the specimen is the same as for the Gram Stain except that a control is not necessary because this is not a differential stain.  [Any technique that is differential distinguishes between two or more types of bacteria.]
2. Gram Stain — uses four reagents and results in distinguishing between bacteria with two types of cell wall structures (gram positive and gram negative).  This is a differential staining technique.
   • The preparation is the same as for the simple stain except that a known gram positive and gram negative are used as controls.
   • The control:  the control is a mixture of Gram + and Gram – bacteria of two shapes.
     • We use Staphylococcus aureus which is a Gram + spherical-shaped (coccoid) bacterium and Escherichia coli [E. coli] which is a Gram - rod-shaped (bacillus) bacterium.
     • Since we use bacteria of different shapes we can mix the two controls and do not have to do a separate + and - control.
   • Cells that are Gram + are purple and those that are Gram - are red (or pink).
3. Negative Stain — uses one stain (Congo Red) which has a negative charge and does not adhere to the negatively charged cells.
   • The background is stained, not the bacterium.  Hence if you use a black stain it looks like a photographic negative.  We don’t use a black stain, we use Congo Red, which is red because it works much better than Nigrosin which is a black negative stain.
     • NO HEAT is applied in the preparation of this stain.  Therefore the cells appear more “natural” — more their normal size and shape.
     • However, this means that they may still be viable and must be treated as such.  All slides with negative stain are disposed of in disinfectant and will be sterilized and recycled for your use.
     • NOTE that for the simple and gram stain, since the specimens are killed by heat fixing they are not to be discarded in the disinfectant.  You may keep them in your slide boxes OR you may clean and reuse them.  If you are going to discard the slides do so in the glass discard barrel NOT in the buckets under the hood.

All stained slides are to be viewed at 1000x total magnification.  Lower magnifications are not sufficient to really see and describe the bacteria accurately.

Oil is applied directly to the dry stained slide.  No cover slip is used.

BE SURE TO READ THE COMIC BOOK ON STAINING — IT GOES THOUGH THE PROCEDURE IN DETAIL.

Practice is the key to successful staining technique.

In lab do these stains:

1. One simple stain
2. One negative stain
3. The following gram stains

• Do a gram stain from your thioglycollate broth from the soil lab. What do you expect to see here?
• Create reference slides (slides you will keep to refer to as you do your unknown) of the following:
  • Staphylococcus [gram + cocci in clusters]
  • Micrococcus luteus [gram + cocci in a sarcinate arrangement]
  • Streptococcus faecalis [gram + cocci in a string; note these sometimes look like slightly elongated cocci]
  • Bacillus subtilis or B. megaterium (whichever one we have out)
    • fairly large gram + rods; can form endospores
    • older cultures of Bacillus can sometimes be “gram variable” which means that some will look gram negative.
  • Eschericia coli (E. coli) [gram - rods]
  • Salmonella sp. [The "sp." means "species" -- I'm not sure what species we have.]  [gram - rods]

  Save your reference slides by dabbing off the oil and placing them in your blue slide box.

• Remember that for each and every gram stain you do, you must include a control mixture.
  • If you wish you can fit three samples on 1 slide:  one control and two other kinds of bacteria in each of two other circles.
  • Exercise 10 Isolation of Bacteria

Exercise 10 Isolation of Bacteria

In order to isolate bacteria you must spread the sample so thinnly that it separates individual cells which can then grow into visible colonies.   This is done in order to make a pure culture which can then be used for identification of the bacteiral species that is isolated.  [See Koch's postulates for the importance of this procedure.]

We use the “Streak Plate Technqiue”.

READ THE COMIC BOOK AS WELL AS YOUR LAB BOOK FOR THIS TECHNIQUE.

Each student will be doing two streak plates using a mixed culture that we provide:

1. One on a general-purpose medium:  Trypticase Soy Agar (TSA)
2. One on a selective medium  [Be sure you know what this means.]
   • Mannitol Salt Agar (MSA)*
   • Eosin Methyleneblue Agar (EMB)*
   • Phenylethylalcohol Agar (PEA)

• *These are also differential media.

These plates will be incubated for 48 hours at 37 degrees C and evaluated in the comeback lab period.

Aseptic technqiue is crutial in this process!  Review your aseptic technqiue.

Be sure to follow the directions exactly.

For what these media are used for and their ingredience see the lab book and the microbiological media information sheet in Vancko Hall.