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

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

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

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.

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”

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

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-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

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

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.

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

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!!!!!!!!!

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

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

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

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!!!!!!!