Specimen Collection and Staining

Question 1

Eukaryotic cells

Answer 1

Contain genetic material within a membrane (nucleus)

Question 2

Prokaryotic cells

Answer 2

Contain genetic material but does not have a nuclear membrane.

Question 3

Is bacteria Eukaryotic or Prokaryotic?

Answer 3

Prokaryotic

Question 4

What does presumptive identification mean?

Answer 4

“Best guess” on what is growing

Question 5

What is the process of identifying bacteria?

Answer 5

1. Collect the specimen
2. Initial Gram stain (Direct or Indirect)
3. Inoculate culture media
4. Incubate for 18-24 hours
5. Check growth. If no growth reincubate and recheck
6. If positive: select colonies and perform gram staining and continue with ID

Question 6

What is used to collect samples from hollow organs and external lesions?

Answer 6

Aspiration (bladder or pustules)

Question 7

What samples need to be processed immediately?

Answer 7

Swab samples

Question 8

What should the specimen contain?

Answer 8

The organism causing the problem

Question 9

What should you do to keep yourself safe while working with microbes?

Answer 9

Wear gloves, lab coats, and closed toed shoes

Question 10

Why do we use gram staining?

Answer 10

To categorize bacteria, which helps the veterinarian select treatment

Question 11

What is the first step of the gram staining process

Answer 11

Use a wax pencil to draw a circle on the slide

Question 12

What is the second step of the gram staining process

Answer 12

Place distilled or sterile water and the sample in the wax pencil circle

Question 13

What is the third step of the gram staining process?

Answer 13

Air drying

Question 14

What is the fourth step of the gram staining process

Answer 14

Heat fixing

Question 15

After heat fixing, what do you flood the slide with?

Answer 15

Crystal violet

Question 16

How long do you flood the slide with crystal violet for?

Answer 16

30-60 seconds, but read the package insert for exact time

Question 17

What do you rinse the slide with after letting the crystal violet sit on the slide?

Answer 17

Distilled water

Question 18

What is the next solution that you flood the slide with after you rinse the Crystal violet?

Answer 18

Iodine for 30-60 seconds

Rinse with distilled water after

Question 19

What is the the next solution you flood the gram slide with after rinsing the Iodine?

Answer 19

Decolorizer for 10 seconds until no more color washes off and then rinse with distilled water

Question 20

What is the final solution you rinse the gram stain slide with?

Answer 20

Safranin for 30-60 seconds.

Question 21

The primary stain is usually what?

Answer 21

Crystal violet

Question 22

What is the the mordant stain and what does it usually do?

Answer 22

Gram’s Iodine solution and it fixes dyes to the cell wall

Question 23

What is the Decolorizer?

Answer 23

95% ethanol or acetone

Question 24

What is the Counterstain?

Answer 24

Basic fuchsin or safranin

Question 25

Gram positive bacteria contain what percentage of peptidoglycans in the cell wall?

Answer 25

60-90%

Question 26

What color do gram positive bacteria turn?

Answer 26

Purple.

Question 27

Why do gram positive bacteria turn purple?

Answer 27

Because of the high number of peptidoglycans

Question 28

Gram negative bacteria contain what percentage of peptidoglycans in the cell wall?

Answer 28

10-20%

Question 29

What color do gram negative bacteria turn?

Answer 29

Pink

Question 30

Why do gram negative bacteria turn pink?

Answer 30

Because of the low number of peptidoglycans and they lose the crystal- violet with the decolorizer

Question 31

What does Gram variable mean?

Answer 31

Gram variable implies that the bacteria will stain both purple and pink but have the same morphology

Question 32

Is Gram variable the same as a mixed colony?

Answer 32

No. Mixed colony can have cocci and bacilli together and gram variable has the same morphology

Question 33

Why does gram variable occur?

Answer 33

``` Excessive decolorization Overly thick smear Excessive heat fixation Old cultures Poor quality stain (Iodine or Mordant being exposed to light) ```

Question 34

what test is used to confirm gram status when a gram variable occurs?

Answer 34

Potassium Hydroxide Test (KOH test)

Question 35

How do you perform a KOH test?

Answer 35

1. A loopful of 3% KOH solution placed onto a slide 2. Generous quantity of surface growth from culture & transferred to slide 3. Stir with a loop

Question 36

What will develop a mucoid appearance and produce a sticky strand when lifted?

Answer 36

Gram negative bacteria

Question 37

What stain is used to ID acid-fast organisms?

Answer 37

Ziehl-Neelsen

Question 38

What acid-fast organisms does the Ziehl-Neelsen identify?

Answer 38

Mycobacterium and Nocardia

Question 39

What are the three parts of the Ziehl-Neelsen?

Answer 39

Primary Stain (RED) Acid-Alcohol decolorizer Counterstain (BLUE)

Question 40

What is the Giemsa stain used to detect?

Answer 40

Spirochetes and rickettsiae Capsule of Bacillus anthracis Morphology of Dermatophilus congolensis

Question 41

Can you use Diff-Quik to stain the slide if you do not have any of the other stains?

Answer 41

Yes but all of the bacteria will be purple

Question 42

What are the common quality controls you can purchase?

Answer 42

Staphylococcus aureus Streptococcus spp Escherichia coli

Question 43

Why do we stain?

Answer 43

Because morphology helps with ID

Question 44

What morphology characteristics help with ID?

Answer 44

Size Shape Arrangement Presence of spores

Question 45

Bacilli

Answer 45

Shaped like rods/cylinders

Question 46

Cocci

Answer 46

Spherical cells

Question 47

Spirochetes

Answer 47

Occur singly and can be loose spirals, tight spirals and comma-shaped spirals

Question 48

Pleomorphic

Answer 48

Shapes ranging from cocci to rods

Question 49

Single Arrangement

Answer 49

Spirilla and most Bacilli

Question 50

Arrangement in pairs

Answer 50

Streptococcus pneumoniae (diplococcus)

Question 51

Chain Arrangement

Answer 51

Short/Long Streptococcus spp

Question 52

Tetrads Arrangement

Answer 52

"Quads"

Question 53

Cluster Arrangement

Answer 53

Staphylococcus aureus (grapelike)

Question 54

Palisades

Answer 54

"Chinese letter" pattern | Corynebacterium spp

Question 55

What do some genera bacteria produce?

Answer 55

Endospores

Question 56

Endospores

Answer 56

Resistant to heat, desiccation, chemicals and radiation