Acid Fast And Endospore Staining

Front 1

Acid fast staining is required to visualize organisms such as

Back 1

the Mycobacterium species.

Front 2

Why can simple stains and gram stains not be used on mycobacteria

Back 2

Because of the impenetrable, waxy lipid cell wall of the mycobacteria

Front 3

the waxy lipid of the mycobacteria is called

Back 3

mycolic acid

Front 4

Mycobacteria is named after this lipid.

Back 4

mycolic acid

Front 5

Carbolfuchsin contains

Back 5

the dye fuchsin and carbolic acid

Front 6

When the mycobacteria are heated in the presence of carbolfuchsin, the heat and the acid do what

Back 6

promote the penetration of the dye into the acid-fast cell wall.

Front 7

Why will decolorizer not wash acid fast organisms

Back 7

carbolfuchsin is soluble in lipid and not in the alcohol

Front 8

Non-acid fast organisms such as E. coli or any of the other bugs we used for a Gram stain would easily decolorize in acid alcohol and take up the counter stain of

Back 8

methylene blue

Front 9

When a slide is counterstained with methylene blue, which organisms will take up the stain

Back 9

non-acid fast organisms will take up the blue dye.

Front 10

Why is it important to use a clean slide for acid fast staining

Back 10

the waxy mycobacteria will begin releasing from the slide during staining and washing

Front 11

Why are a clean slide, and proper heat fixing of the bacterial smears very important in acid fast staining

Back 11

the waxy organisms can release from the slide

Front 12

Gram stains on mycobacterium can appear how

Back 12

can be mistaken as a truly gram sensitive organism

Front 13

mycobacteria appear how when grown on agar

Back 13

very distinctive, dry, waxy appearance on agar

Front 14

Endospores are

Back 14

highly resistant, dormant forms of bacteria which can survive extremely long periods of time in harsh conditions.

Front 15

Members of the genus, Bacillus, are all Gram _____

Back 15

Gram positive endospore producing cells

Front 16

All members of the genus Bacillus have what defining characteristics

Back 16

all are Gram Positive and endospore producing

Front 17

After growth for a couple of days in a test tube, a Bacillus culture will exhaust its supply of nutrients and will actively produce

Back 17

endospores in order to survive.

Front 18

Members of the genus, Clostridium, are

Back 18

anaerobic, Gram positive bacilli.

Front 19

In addition to the depletion of nutrients, Clostridium species will respond to the presence of oxygen as a

Back 19

negative signal or hostile environment.

Front 20

Clostridiums response to oxygen as a negative signal or hostile environment will cause the organism to

Back 20

begin to sporulate.

Front 21

The Clostridium used for the endospore stain will be grown in a

Back 21

candle jar.

Front 22

How is clostridium grown in a candle jar

Back 22

The tubes are placed in a jar with a lit candle, and the lid of the jar is screwed on tightly. The candle consumes available oxygen and flame extinguishes.

Front 23

Why is it important to create a good environment for the clostridium to grow using the candle jar technique

Back 23

in order to prevent sporulation

Front 24

The boiling of the malachite green with the bacteria in an endospore stain facilitates

Back 24

the penetration of the green stain into the endospores

Front 25

Following endospore staining with malachite green, bacilli are counterstained with

Back 25

safranin

Front 26

Endospore staining of bacilli should look like

Back 26

pink bacilli with green endospore within them

Front 27

The Acid fast stain is a

Back 27

differential stain

Front 28

The acid fast differential stain allows differentiation between what

Back 28

differentiate between acid fast and non-acid fast bacteria.

Front 29

Acid fast refers to

Back 29

the ability of certain cells to retain a lipid soluble dye even when washed with acid.

Front 30

Acid fast bacteria include

Back 30

the Mycobacterium and Nocardia genuses which have a waxy lipid called mycolic acid as a component of their cell walls.

Front 31

Non-acid fast bacteria lack this lipid and decolorize when washed with acid

Back 31

mycolic acid

Front 32

The endospore stain is a special stain used to identify

Back 32

bacteria capable of producing endospores.

Front 33

Endospores resist staining with normal techniques and must be _____ in order to drive the dye into the endospore

Back 33

heated in the presence of dye

Front 34

E. coli is a

Back 34

non-acid fast, Gram negative bacillus.

Front 35

Mycobacterium species are

Back 35

acid fast bacilli.

Front 36

C. pseudodiphtheriticum is a

Back 36

pleomorphic bacillus, non-endospore producing and partially acid fast.

Front 37

Bacillus and Clostridium species DO or DO NOT produce endospores

Back 37

Both are endospore producers

Front 38

Cultures were grown at 37oC with loose caps with the exception of ______ which is an anaerobe.

Back 38

Clostridium

Front 39

The sporulating organisms were grown at least __ hours to ensure they are running low on nutrients and have begun to sporulate

Back 39

72

Front 40

The anaerobe was transferred out of the anaerobic chamber a couple of days in advance to promote sporulation in the presence of _________

Back 40

oxygen.

Front 41

Acid Fast Stain Procedures

Back 41

1. Generate smear on extra clean slide.
2. Air dry!
3. Heat fix!
4. Place over steaming beaker of tap water. Add filter paper cut to size of smear. Saturate smear with Carbolfuchsin (a basic, lipid soluble dye) and heat for 5 minutes, keeping paper moist so it doesn’t stick to smear.
5. Remove paper with forceps and wash with dH2O for 30 seconds.
6. Decolorize with acid-alcohol for 15 seconds.
7. Rinse with dH2O.
8. Flood smear with methylene blue for 2 minutes.
9. Rinse with dH2O.
10. Blot dry. Wash back of slide with an alcohol soaked Kimwipe.
11. Observe using immersion oil.

Front 42

E. coli is non-acid fast and will be what color following the acid fast procedure

Back 42

blue

Front 43

Mycobacterium will be what color following the acid-fast procedure

Back 43

fuchsia (pink) and is acid-fast.

Front 44

Endospore Staining Procedures

Back 44

1. Generate smear on extra clean slide.
2. Air dry!
3. Heat fix!
4. Place over steaming beaker of tap water. Add filter paper cut to size of smear. Saturate smear with malachite green and heat for 5 minutes, keeping paper moist.
5. Remove paper with forceps and rinse with dH2O for 30 seconds.
6. Flood smear with safranin for 2-3 minutes.
7. Rinse with dH2O.
8. Blot dry. Wash back of slide with an alcohol soaked Kimwipe.
9. Observe using immersion oil.

Front 45

Endospores will take up the _____ ______ and will be pale teal green in color

Back 45

Malachite Green

Front 46

The endospore producing parent bacilli will be stained by _______and will be what color

Back 46

safranin and will be pink