Microbio lab Unknown bacteria identification

Question 1

labeling the Mac Conkey agar plate

how do you label the plate?

Answer 1

The plate must be labeled with as much inormation as known at this time bc we are working with unknown organisms.
Place label on the bottom (agar side)

Accurate and appropriate labels are important in case of accidental expsures.

Question 2

steps of lactose fermentation testing

Answer 2

1. label MacConkey agar plate on the bottom
2. Turn on bunsen burner
3. select loop and sterilize it
4. pick up unknown culture, remove cap, heat mouth in bunsen burner to reduce contamination
5. Insert loop into tube to get bacterial sample
6. briefly heat mouth of tube, recap, return to tube rack
7. Inoculate MacConkey agar plate with loop
8. heat loop, return to holder
9. incubate button to grow bacteria on Macconkey agar plate at 37C for 24 hrs.
10. Review Lactose fermentation test description and interpret results

Question 3

MacConkey agar helps detect lactose fermentation into acidic products and is used to classify bacteria based on this result for an unknown organism. Here are the results of your MacConkey agar growth. What can you conclude?

Answer 3

The MacConkey agar test shows the unknown bacterium is positive for lactose fermentation.

on the picture: plate is pink and dark purple bacterium shows in dots. I thought acid was yellow.

Question 4

Gram staining (test)

describe the process

Answer 4

The Gram stain is usually the first step in the identification of an isolated unknown organism and probably the most important stain used in microbiology. The Gram stain is a differential stain - meaning it differentiates by color two major groups of bacteria: the gram-positive and the gram-negative bacteria. This is based on their cell wall location and chemical composition. The Gram stain technique involves the use of dyes/stains, a mordant, and a decolorizer.
Crystal violet, the primary stain, stains all of the cells on the slide purple as the dye binds to a cell wall component known as peptidoglycan.

The slide is rinsed with water and then the smear is covered with

Gram’s iodine (mordant) to fix color into the cell or intensify color.

The next step is decolorization with 95% ethanol, washing out the iodine–crystal violet complex in gram-negative cells, but does not affect the color in gram-positive cells.
After the decolorization step, gram-negative cells are colorless and gram-positive cells are purple.
The final step is the counterstain,
safranin to add a contrast color to the “invisible” gram-negative cells. After staining, gram-positive bacterial cells will appear purple, and gram-negative cells will appear red.

Question 5

Oxidase test

to test for the presence of this enzyme

Answer 5

To test for the presence of this enzyme, a reagent called oxidase reagent is added to the cells of interest.
The reagent is a chemical indicator that is colorless when reduced and dark blue-to-purple when oxidized. The oxidase reagent will donate electrons to cytochrome c oxidase if it is present. By donating electrons, the reagent is oxidized and turns dark blue-to-purple. This color change is an indication that the organism produces cytochrome c oxidase as part of its electron transport chain. This procedure is used to distinguish among the gram-negative rods.

Question 6

LACTOSE FERMENTATION

CARBOHYDRATE METABOLISM

Answer 6

Carbohydrate metabolism pathways vary throughout gram-negative bacteria. To observe the ability of the unknown bacterium to ferment lactose, a test known as a MacConkey agar plate is used. MacConkey agar ingredients make it a selective-differential medium, allowing you to select for specific bacteria that are able to grow and simultaneously see differences in those bacteria. MacConkey agar contains bile salts and the dye crystal violet to make the medium selective by inhibiting the growth of gram-positive bacteria but allowing many gram-negatives to grow. In addition, MacConkey agar contains the sugar lactose and the pH indicator neutral red, which allows for differentiation between non-lactose fermenters and lactose fermenters, based on color changes. During the fermentation of lactose, acid is produced and released into the medium. In response to the lower pH, neutral red turns from pink to red. The color change distinguishes lactose-fermenting bacteria from other gram-negative bacteria.

Colonies of lactose fermenters will appear purple-to-pink. Non-lactose-fermenting gram-negative organisms will be clear or uncolored.

Question 7

METHYL RED TESTING

ITEMIZE METHYL RED TESTING STEPS

Answer 7

1. turn on bunsen burner
2. select loop, sterilize
3. pick up bacterial culture, cap, heat tube’s mouth
4. insert loop, get bacteria
5. heat mouth of tube, recap, return to rack
6. pick up MR-VP broth tube, remove cap, heat mouth,
7. insert loop with sample on it from the other tube
8. heat mouth of MR VP broth tube, recap, return tube to rack
9. sterilize loop
10. Incubate to grow bacteria at 37C for 24 hrs.
11. add several drops of methyl red reagent to incubated MR VP broth tube
12. Review Methyl Red Test description if needed, interpret results
13. select next logical testing step