4/12/2010Hewitt-Trussville High School

Walters

Gram Stain Lab!!!!

How are people infected?Contact with infected body fluids.

Mucous from a cough or sneezeBloodFeces

Contact with the air, water, or food borne infectious agent.

Contact with a contaminated surface.Door knobTelephone

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:MosquitoesFleasTicks

Heart Disease and InfectionsRheumatic Fever

Begins as Strep throat but toxin produced by bacteria damages heart valves.

Chronic Inflammation and atherosclerosisLong term exposure to the immune products

made in response to infections may lead to hardening of the arteries.

NotesMost bacteria are colorless

Needed a way to view them under a microscope

In 1894, Christian Gram (Danish) discovered a method to stain bacteriaBased on bacteria that have a cell wall and

those that do not!Turns purple if microorganism can retain crystal

violet stainTurns red if it cannot retain violet stain95% Ethyl Alcohol is the “decolorizing agent”

Gram Stain TechniqueGram Negative Bacteria

After adding crystal violet stain and alcohol, these bacteria are decolorizedNo cell wall to hold the stain

After the violet stain, a counterstain is used to turn the bacteria pink (safranin)

Gram Positive BacteriaThe cell wall holds the crystal violet stain

Bacteria Shapes

Bacteria have three basic shapes:

cocci (spherical)

bacilli (rectangular)

spirochete (spiral)

http://www.mansfield.ohio-state.edu/~sabedon/biol2010.htm#illustration_cocci

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.

Gram + Vs. Gram -

Gram positive versus gram negative

Fig. 4.14

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

Gram Positive

Gram Negative

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.

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.

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.

Can You Diagnose These Patients?

A B C

D E

Can You Diagnose These Patients?

- bacilli + cocci + bacilli

- cocci + cocci

DifficultyCarefully 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.

Step 1Obtain the following:

ApronGlovesSafety GlassesRed bucket

1 tube of bacteria1 inoculation loops1 microscope slidesCrystal violet stainSafarain StainIodine Stain95% Ethyl Alcohol

Step 2 - 72. 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 is3. Heat the inoculation loop using the Bunsen burner4. Flame the mouth of the culture tube (bacteria) using

the burner5. 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

6. Heat the mouth of the culture AGAIN and replace the cap

7. Allow the slide to dry!!!!!!!!!

Steps 7 - 107. Using the forceps…

Pass the microscope slide through the Bunsen burner flame three times SMEAR SIDE MUST BE SIDE UP AWAY FROM THE

FLAME!!!!

8. Allow the slide to cool9. 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

10. Rinse the slide with water from the water bottle

Steps 11 - 1511. 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

12. Decolorize with 95% ethyl alcohol Allow the alcohol to remain on the slide 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

13. Rinse the slide with water again14. Add 1 drop counterstain Safranin and 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

15. Rinse the slide with water again

Steps 16 - 1716. Blot the microscope slide dry with a paper

towel17. Get the slide ready to put under the

microscope.

Clean-upPlace inoculation loops into the bleachReplace all of the stains into the red bucketReplace the sharpie and droppers back into

the red bucketCarefully replace the bacteria into the red

bucket.Replace the aprons and glasses into the

appropriate areas.Throw away glovesWash hands with soap and water!!!!!!!