Microbiology Lab Class Notes

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Shakira Jarvis
Microbiology Lab Assignment

Laboratory Assignment Outline

1. Check in & The Microscope
a. Review of proper lab etiquette.
i. Review laboratory syllabus and
b. Review of the Parts of a Microscope
ii. Review of lab exercises about different types of Microscopes 2. Survey of Microorganisms
c. Viewing, drawing, and describing several types of fungi, algae, and Bacteria iii. Chlamydomonas
iv. Spirogyra
v. Penicillum mycelium condidiophores
vi. Cladophora
vii. Volvox
viii. Paramecium caudatum
ix. Escherichia coli
x. Bacillus subtilis
xi. Staphylococcus aureus
xii. Aspergillus condidiophores
xiii. Amoeba Proteus
xiv. Budding Yeast
xv. Diatoms ( Marine)
xvi. Peziza Apothecium
xvii. Coprinus Mushroom
xviii. Tryanosoma lewisi
xix. Plasmodium malariae
3. Aseptic Technique, Smear Preparation, Simple & Negative Staining d. Aseptic Technique
xx. Work area disinfection
xxi. Flaming loops/ needles before and after use
xxii. Flaming the top of tubes
xxiii. Washing hands before and after lab
e. Smear Preparation
xxiv. Can be prepared from either broth of agar plate 1. Broth
a. No water added
b. Flame loop every time it needs to be reintroduced to the culture c. Spread all over slide
d. Let dry & heat fix
2. Agar
e. Add drop of water to CLEAN slide
f. Flame loop, let cool, get loop full of bacteria g. Spread bacteria through out drop of water h. Spread all over slide
i. Let air dry then Heat fix
f. Simple Stain –
xxv. one dye ( Methylene blue) stains the organism xxvi. can only determine morphology and arrangement xxvii. Procedure
3. Prepare smear
4. Add Methylene blue ( 30 sec. )
5. Wash w/ water
6. Blot dry
7. View
g. Negative Stain –
xxviii. One dye ( Nigrosin = [acidic dye w/ negative charge] ) does not stain the organism [also negatively charged], but rather the environment xxix. Can determine size.
xxx. Procedure
8. Prepare smear
j. DO NOT HEAT FIX!
9. Add Nigrosin
10. Let air dry
11. View
4. Differential Stains (Gram Stain, Acid Fast Stain, Endospore Stain) h. Differential Stains – Stains that classify the differences in two organisms i. Gram Stain – Differentiates between organisms with a thick layer of peptidoglycan in their cell wall versus organisms with a thin layer of peptidoglycan. xxxi. Procedure

12. Prepare smear
13. Stain with primary stain Crystal Violet for 30 seconds 14. Wash w/ water
15. Add mordant, Gram’s Iodine for 1 minutes
16. Wash w/ water
17. Add decolorizer, 95% ethanol for 10 to 15 seconds 18. Wash w/ water
19. Add secondary stain Safranin for 1 minute
20. Wash with water
21. Blot dry
22. View
xxxii. Results
23. Gram Negative – Appears pink ( ex. Escherichia coli) 24. Gram Positive – Appears purple ( ex. Staphylococcus aureus) j. Endospore Stain – Differentiates betweens endospores and vegetative cells in bacteria that form spores ( ex. B. subtilis) xxxiii. Procedure ( Schaeffer fulton Method)

25. Prepare smear
26. Put slide of steam and add Primary stain (Malachite Green) to slide via a piece of paper towel for 5 minutes...
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