Bacterial smear for slant
put bacteria on slide, add water, air dry, heat fix
bacterial smear for broth
put bacteria on slide, air dry, heat fix
Why are thick or dense smears less likely to provide a good smear preparation for microscope evaluation?
Doesn't allow enough light through
Why is it essential that smears be air-dried? Why can't they be gently heated over a flame to speed up the drying process?
Doesn't kill the bacteria. To help the bacteria keep shape.
Why should you be careful not to overheat the smear during the heat-fixing process?
Kill the bacteria. Burn things off. Distort the shape.
Why do you think the presence of grease or dirt on a glass slide will result in a poor smear preparation?
Oil from fingers prevent sticking to slide. Dust will look microbes on the slide.
simple staining
stain, heat fix, blot
dyes used to simple stain
Methylene Blue, Crystal Violet, and Carbon Fuchsin
Why are dyes more effective for bacterial staining than acidic dyes?
Bacteria have negative charge and basic dyes have a positive charge so they attract from a better bond then the acidic dyes which are also a negative charge.
Can simple staining techniques be used to identify more than the morphological characteristics of microorganisms?
No only got morphology. Nothing in the stain would allow us to see anything but shape and arrangement.
During the performance of the simple staining procedure, you failed to heat fix your E. coli smear preparation, Upon microscopic examination, how would you expect this slide to differ from the correctly prepared slides?
We will not see anything because we washed off the bacteria when we rinsed the slide off.
Gram Straining Procedure
Gently flood smear with Crystal Violet stand for 1 min, gently rinse, gently flood smear with Gram's Iodine stand for 1 min, gently rinse, add 95% Ethyl Alcohol until smear is colorless, gently rinse, cover smear with Safranin stand for 45 seconds, gently
What are the advantages of differential staining procedures over the simple staining technique?
Allows us to see more than just morphology, ex: gram stain shows which bacteria are gram positive or negative
Cite the purpose of each of the following reagents in a differential staining procedure
Crystal Violent: add color
Iodine: hold color in cell
Alcohol: remove the colors from gram negative because amount of peptidoglycan
Safranin: color the cells that loss color aka gram negative cells
Why is it essential that the primary stain and the counter stain be of contrasting colors?
So we can see the difference between the gram positive and negative cells
Which is the most crucial step in the performance of the gram staining procedures?
Decolorization because you can get the wrong result if you add too much or too little
Because of a snowstorm, your regular laboratory session was canceled and the Gram staining procedure was performed on cultures incubated for a longer period of time. Examination of the strained B. cereus slides revealed a great deal of color variability,
some gram positive bacteria will lose some ability to show true/correct color as they age
Citrate Test
Test for presence of Citrate
SIM procedure
Inoculate the deep agar. Positive if it turns black
Urease Test
Test for presence of Urease
Urease Procedure
Inoculate the broth. Positive if turns from peach to pink
Fermentation Test procedure and positive result
Inoculate the three test tubes. Positive if they produce air bubbles in inverted test tubes producing gas. Positive if they turn bright yellow producing acid.
Fermentation test
Phenol Red Sucrose, Phenol Red Lactose Broth, Phenol Red Dextrose Broth; checking to see if they produce gas and/or acids
SIM Test
Test for production of Hydrogen sulfide
Indole Test
Test for the ability to use amino acid trytophan
Citrate Procedure
Inoculate the slant. Positive if turns dark blue.
Indole Procedure
Take test tube from Hydrogen Sulfide test add a couple drops Kovak's reagent. Positive if turns cherry red.
Starch Hydrolysis Test
Test for the ability to hydrolyze starch
Starch Hydrolysis Procedure
Smear on plate. Flood with Gram's Iodine. Positive if they are clear spots in the plate.
Catalase Test
Test for production of of Catalase.
Catalase Procedure
Inoculate the slant. Positive if it produces bubbles.