Download Gram Stain Lab!!!!

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
 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 gramnegative bacteria.
 Therefore G+ can hold the violet dye, whereas Gcan’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
D
E
C
Can You Diagnose These Patients?
- bacilli
+ cocci
- cocci
+ cocci
+ bacilli
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
2.
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


3.
4.
5.
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



6.
7.
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
7.
Using the forceps…
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
9. Add a drop of the crystal violet stain to the bacteria
8.
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 - 15
11. 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 again
14. 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 - 17
16. Blot the microscope slide dry with a paper towel
17. 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!!!!!!!