1. 4/12/2010 Hewitt-Trussville High School Walters
Gram Stain Lab!!!!
4/12/2010
Hewitt-Trussville High School
Walters

2. How are people infected?
Contact with infected body fluids.
Mucous from a cough or sneeze
Blood
Feces
Contact with the air, water, or food borne infectious agent.
Contact with a contaminated surface.
Door knob
Telephone

3. How are the diseases spread?
From person-to-person.
Colds, Flu, Small pox, Polio
From animal-to-person.
Rabies, Brucellosis, Cat Scratch Fever
Through contaminated food, soil, water, or other material.
By disease vectors including:
Mosquitoes
Fleas
Ticks

4. Heart Disease and Infections
Rheumatic Fever
Begins as Strep throat but toxin produced by bacteria damages heart valves.
Chronic Inflammation and atherosclerosis
Long term exposure to the immune products made in response to infections may lead to hardening of the arteries.

5. Notes Most bacteria are colorless
Needed a way to view them under a microscope
In 1894, Christian Gram (Danish) discovered a method to stain bacteria
Based on bacteria that have a cell wall and those that do not!
Turns purple if microorganism can retain crystal violet stain
Turns red if it cannot retain violet stain
95% Ethyl Alcohol is the “decolorizing agent”

6. Gram Stain Technique Gram Negative Bacteria Gram Positive Bacteria
After adding crystal violet stain and alcohol, these bacteria are decolorized
No cell wall to hold the stain
After the violet stain, a counterstain is used to turn the bacteria pink (safranin)
Gram Positive Bacteria
The cell wall holds the crystal violet stain

7. Bacteria Shapes Bacteria have three basic shapes: cocci (spherical)
bacilli (rectangular)
spirochete (spiral)

8. Gram Stains
Based on the ability of bacteria cell wall to retain the violet dye during staining.
Bacteria are either Gram positive and appear purple or Gram negative and appear pink.
The reaction to the Gram stain is dependent on the structure of the bacterial cell wall.
Gram-positive microorganisms have a higher peptidoglycan and lower lipid content than gram- negative bacteria.
Therefore G+ can hold the violet dye, whereas G- can’t; they get re-stained pink.

9. Gram Vs. Gram -

10. Gram positive versus gram negative

11. Fig. 4.14

12. Gram-Positive vs. Gram-Negative
Gram summary
Gram-Positive vs. Gram-Negative
Thick peptidoglycan
Teichoic acids
In acid-fast cells, contains mycolic acid
Thin peptidoglycan
No teichoic acids
Outer membrane

13. Gram Positive

14. Gram Negative

15. What’s The Difference?
The effectiveness of antibiotics is dependent on the mechanism of action of the drug and the structure of the bacteria.
Ex. penicillin is very effective against Gram positive.
It acts by inhibiting the formation of the peptidoglycan linkages found in the cell walls of Gram positive bacteria.
It has little effect on the formation of the cell walls of Gram negative bacteria, and hence has little effectiveness in treating infections caused by Gram negative bacteria.

16. What’s the difference?
Alternatively, the antibiotic streptomycin works by binding to the 16S subunit of the ribosome and inhibiting protein production in the bacteria.
Streptomycin is very effective against Gram negative bacteria but has limited effectiveness against Gram positive bacteria.
A broad spectrum antibiotic is effective against both Gram positive and negative bacteria.
Ex. Tetracycline is effective against many different types of bacteria.

17. Physician’s Prescription
When a patient is diagnosed with a bacterial infection, the physician will often prescribe a broad spectrum antibiotic or an antibiotic commonly used for the particular type of infection.
If the patient’s health does not improve, then the physician may take a sample of the bacteria from the infection site and test several different antibiotics to find the best one to use.

18. Can You Diagnose These Patients?
A B C
D E

19. Can You Diagnose These Patients?
- bacilli + cocci + bacilli
- cocci cocci

21. Difficulty Carefully handle the tube of bacteria.
DO NOT MAKE YOUR BACTERIA SMEARS TOO THICK!!!
Do not overheat your microscope slide.
It will “cook” and remove the bacteria from the slide
Allow the appropriate amount of time for the stains to react on the slide.

22. Step 1 Obtain the following: Apron Gloves Safety Glasses Red bucket
1 tube of bacteria
1 inoculation loops
1 microscope slides
Crystal violet stain
Safarain Stain
Iodine Stain
95% Ethyl Alcohol

23. Step 2 - 7 Place 1 drop of water IN THE MIDDLE of a clean slide
Use the small dropper
Using a sharpie, mark your name and side where the smear is
Heat the inoculation loop using the Bunsen burner
Flame the mouth of the culture tube (bacteria) using the burner
Using the loop, remove a SMALL/TINY amount of the bacteria (You should barely see anything on the loop)
Mix the bacteria and water on the microscope slide
Use a CIRCULAR MOTION to mix water and bacteria
Should appear as a thin, faintly cloudy, film
Heat the mouth of the culture AGAIN and replace the cap
Allow the slide to dry!!!!!!!!!

24. Steps 7 - 10 Using the forceps… Allow the slide to cool
Pass the microscope slide through the Bunsen burner flame three times
SMEAR SIDE MUST BE SIDE UP AWAY FROM THE FLAME!!!!
Allow the slide to cool
Add a drop of the crystal violet stain to the bacteria
Allow it to react for 60 seconds.
Place slide at a 45 degree angle
Drop at the top of the slide, and allow the stain to run down the slide
Rinse the slide with water from the water bottle

25. Steps 11 - 15 Add 1 drop of iodine stain to the slide
Allow it to react for 60 seconds
Place slide at a 45 degree angle
Drop at the top of the slide, and allow the stain to run down the slide
Decolorize with 95% ethyl alcohol
Allow the alcohol to remain on the slide for 60 seconds
Rinse the slide with water again
Add 1 drop counterstain Safranin and allow it to react for 60 seconds

26. Steps 16 - 17 Blot the microscope slide dry with a paper towel
Get the slide ready to put under the microscope.

27. Clean-up Place inoculation loops into the bleach
Replace all of the stains into the red bucket
Replace the sharpie and droppers back into the red bucket
Carefully replace the bacteria into the red bucket.
Replace the aprons and glasses into the appropriate areas.
Throw away gloves
Wash hands with soap and water!!!!!!!