Lab Midterm

Steps for Gram stain (remember to rinse between each step)

1. Add crystal violet (primary stain). This stain binds to the peptidoglycan of
the cell wall.
2. Add gram iodine (mordant) - makes the crystal violet insoluble.
3. Ethanol-acetone (decolorizes) dissolves outer membrane and washes
uncomplexed primary stain
4. Safranin- counterstain (stains gram negative) gram positive is already
stained from the crystal violet.
5. End results gram positive is purple and gram negative is red
6. Gram stain is a differential stain.
7. Gram staining has something to do with the cell wall. Gram positive has a
thick membrane and gram negative has an outer membrane.

Capsule stain
Purpose to determine if a capsule is present
Remember to no high fixing. Let it air dry

Step Reagent Function Gram + Gram -

Primary Stain Crystal Violet - 20 sec. Binds to peptidoglycan of cell wall purple purple

Rinse Water - 2 sec. Removes excess crystal violet purple purple

Mordant Grams iodine - 1 min. Makes crytal voilet insoluble purple purple

Rinse Water - 2 sec. Removes excess iodine purple purple

Decolorizer 75% ethanol + 25% acetone mix - 10 sec. Dissolves outer membrane and washes uncomplexed primary stain purple clear

Rinse Water - 2 sec. Stops decolorizer purple clear

Counter Stain Safranin - 1 min. stains gram - (b/c gram + already colored will not take color) purple red

Rinse Water - 2 sec. Removes excess safranin purple red

1. Bacterial endospores are not produced by:

A. Staphylococcus**
B. Sporosarcina
 C. Bacillus
D. Clostridum

2. If during the gram stain procedure, the bacterial cells were viewed
immediately after crystal violet was applied, Gram positive cells would be
purple but Gram negative cells would be colorless. FALSE

3. Iodine is the decolorize in the gram stain. FALSE

4. The cell envelope of Gram-Positive bacteria has two layers a thick cell wall
and the cell membrane. TRUE

5. Which type of plating may be used for isolation only? STREAK

6. Culture media that contains various substances whose precise chemical

composition are unknown. COMPLEX MEDIA

7. Culture media that allows you to separate even closely related group of
microorganisms. DIFFERENTIAL MEDIA

8. If the gram iodine step were omitted in the gram procedure, what color
would you expect gram postive bacteria and gram negative bacteria to stain?
GRAM POSTIVE= PINK, GRAM NEGATVE= PINK

9. During staining, the smear is heat-fixed in order to ________________________.

a kill the organism so that dyes will penetrate.
b attach the organism firmly to the slide
c kill the organism and attach the organism firmly to the slide*
d neither kill the organism nor attach the organism firmly to the slide

10. The colorization of bacteria with more than one dye solution to
distinguish between microbial cell types is called a _________.
A.wet mount
B.simple stain*
C.differential stain
D.structural stain
E. hanging drop
10. a microbiologist inoculates Staphylococcus auerus into a culture medium.
Following incubation, both staphylococcus auerus and staphyloccous
 epidermidis are determined to be growing on this culture. What is the most
likely explnatation? THE CULTURE IS CONTAIMINATON.

11. which structure protects bacteria from being phagocytized by white blood
cells? CAPSULE

12. Which order below reflects the correct procedure for gram staining?
CRYSTAL VIOLET-IODINE, ALCOHOL/ ACETONE, SAFARIN

13. During the gram stain___ cells decolorize when the alcohol is applied. GRAM
NEGATIVE

14. A bacterial genus that has waxy mycolic acid in the cell wall is.
MYCOBACTERIUM.

15. The difference in cell wall structure of mycobacterium and nocardia

compared to the typical gram positive bacterial cell wall structure is:
PREDOMINACE OF UNIQUE WAXY LIPIDS

16. The___ stain is used to stain and differentiate mycobacterium and nocardia
from other bacteria. ACID FAST

17. Cell walls are not found on typical cells of PROTOZOA.

18. In the electron microscope, a stream of_____ is employed as a sources of

illumination instead of visible light. ELECTRON