EXERCISE 10

Question 1

Basic Flow of Laboratory Diagnosis of Infection

Answer 1

1. Gross examination of the specimen
2. Direct microscopic examination of patient specimens for the presence of etiologic agents
3. Growth and cultivation of agents from the same specimens
4. Analysis of the cultivated organisms

Question 2

to note important specimen characteristics that are helpful during sample processing and result reporting

Answer 2

Gross examination of the specimen

Question 3

should be located and sampled for culture and direct examination

Answer 3

areas with blood or mucus

Question 4

the stool should be examined for evidence of [?] (i.e., chalky white color), which would preclude parasitology examination

Answer 4

barium

Question 5

notations should be made on the [?] regarding the status of the specimen (e.g., bloody, cloudy, clotted)

Answer 5

handwritten or electronic work card

Question 6

allows assessment of the quality of the specimen

Answer 6

Direct microscopic examination of patient specimens for the presence of etiologic agents

Question 7

provides an early indicator of what may be wrong with the patient

Answer 7

Direct microscopic examination of patient specimens for the presence of etiologic agents

Question 8

help plan workup of the specimen guided by comparing what grows in culture to what was seen on the smear

Answer 8

Direct microscopic examination of patient specimens for the presence of etiologic agents

Question 9

are usually not performed on throat, nasopharyngeal, or stool specimens but are indicated from most other sources due to the presence of abundant normal flora.

Answer 9

Direct examinations

Question 10

to establish their identity and other pertinent characteristics such as susceptibility to antimicrobial agents

Answer 10

Analysis of the cultivated organisms

Question 11

use of a microscope to magnify objects too small to be visualized with the naked eye so that their characteristics are readily observable

Answer 11

Microscopy

Question 12

Applications of Microscopy in Diagnostic Microbiology

1. [?] by direct visualization in patient specimens.
2. [?] of certain organisms by direct visualization in patient specimens.
3. [?] present in the same specimen.
4. Detection of organisms [?] in the laboratory.
5. [?] for the presence of cells indicative of inflammation (i.e., phagocytes) or contamination (i.e., squamous epithelial cells)
6. Determination of the organism’s [?]; bacterial contaminants usually are not present in-patient specimens at sufficiently high numbers (x105 cells/mL) to be seen by light microscopy.
7. Provide [?] about which organisms might be expected to grow, so that appropriate cultivation technique is used.
8. Determine which [?] should be used for the identification and\ characterization of cultivated organisms.
9. Provide a [?] unusual or unexpected laboratory test results.

Answer 12

1. Rapid preliminary organism identification
2. Rapid final identification
3. Detection of different organisms
4. not easily cultivated
5. Evaluation of specimens
6. clinical significance
7. preculture information
8. tests and methods
9. method for investigating

Question 13

A. Direct Examination of Unstained Specimens

Answer 13

1. Saline Mount
2. Hanging-drop procedure
3. Neufeld’s Quellung Reaction
4. Darkfield Examination
5. Phase Contrast Examination

Question 14

To determine the biological activity of microorganisms such as:
• motility
• reactions to certain chemicals
• serologic reactivity in specific antisera

Answer 14

Saline Mount

Question 15

Saline Mount: Material

Answer 15

0.85% Sodium chloride (aqueous)

paraffin-petrolatum mixture (Vaspar)

Question 16

Saline Mount Techniques:
a. Dispense a small quantity of the specimen to be examined into a drop of [?] on a microscopic slide.
b. Overlay a coverslip and examine directly with a [?] of the microscope, closing the diaphragm to reduce the amount of transmitted
light.
c. To prevent drying, ring the coverslip with a small amount of [?] before overlaying the specimen drop on the slide.

Answer 16

a. saline
b. 40x or 100x objective
c. paraffin-petrolatum

Question 17

Hanging-drop procedure: Material

Answer 17

hanging drop slide (thick glass slide with a central
concave well)
physiological saline or water
paraffin-petrolatum mixture

Question 18

same as the saline mount, except there, is less distortion from the weight of the coverslip, and a deeper field of focus into the drop can be achieved.

Generally used for studying the motility of bacteria

Answer 18

Hanging-drop procedure

Question 19

Hanging-drop procedure Technique:

a. A small amount of paraffin-petrolatum mixture is placed around the lip of the well on the [?] of the hanging-drop slide.
b. Cells from a bacterial colony to be examined are placed in the [?], into a small drop of saline or water.
c. The slide is [?] over the coverslip, guiding the drop of bacterial suspension into the center of the well.
d. The slide is carefully brought to an [?] for direct examination under the microscope.

Answer 19

a. undersurface
b. center of the coverslip
c. inverted and pressed
d. upright position

Question 20

Neufeld’s Quellung Reaction: Material

Answer 20

Homologous anticapsular serum

physiologic saline

Question 21

When species of encapsulated bacteria are brought into contact with serum-containing homologous anticapsular antibodies, their capsules undergo a change in refractive index to produce “swelling” that is visible by microscopic examination. The serologic procedure is useful in identifying the various types of Streptococcus pneumoniae and Haemophilus influenzae in biologic fluid or culture

Answer 21

Neufeld’s Quellung Reaction

Question 22

Neufeld’s Quellung Reaction Technique:

a. A loopful of material, such as [?], body fluid, or broth culture, is spread over a 1 cm area in two places on opposite ends of a microscope slide.
b. A loopful of [?] is spread over the area of one of the dried preparations; the opposite area is overlaid with a loopful of saline to serve as a control.
c. Each area is overlaid with a coverslip and examined under the [?] objective of the microscope.
d. Organisms showing a positive reaction appear surrounded by a [?], refractile halo owing to capsular swelling.
e. Compare the test preparation with the [?] where no capsular swelling occurs.

Answer 22

a. emulsified sputum
b. specific anticapsular typing serum
c. 100x
d. ground-glass
e. saline control

Question 23

Darkfield Examination: Material

Answer 23

Compound microscope equipped with a darkfield condenser
Physiologic saline
a paraffin-petroleum mixture

Question 24

To visualize certain delicate microorganisms that are invisible by brightfield optics and stain only with great difficulty; useful in demonstrating spirochetes from suspicious syphilitic chancres for Treponema pallidum

Answer 24

Darkfield Examination

Question 25

Darkfield Examination Technique:
a. The secretion to be examined is obtained from a patient. In the case of a chancre, the [?] is scraped away with a scalpel blade and a small quantity of serous material is placed on a microscope slide.
b. Ring a coverslip with [?] and place over the drop of material.
c. Examine the mount directly under a microscope
fitted with a darkfield condenser with [?] objectives. Spirochetes will appear as motile, bright “corkscrews” against a black background.

Answer 25

a. top crust
b. paraffin-petrolatum mixture
c. 40x or 100 x

Question 26

Limitations: The need for experienced personnel to perform the test and the need for a

Answer 26

darkfield microscope

Question 27

allows observation of viable microorganisms

Answer 27

Phase Contrast Examination

Question 28

not commonly used in most diagnostic microbiology, but it is used to identify medically important fungi grown in culture

Answer 28

Phase Contrast Examination

Question 29

Purpose of SMEAR PREPARATION

To prepare a [?]of bacterial growth on a glass slide (smear) to proceed with further staining for microscopic examination.

Answer 29

thin, uniform film

Question 30

Preparation of smear requires attention to a number of details that help prevent [?] and [?]. Each step has an important reason, and each should be followed carefully as a prerequisite to successful work.

Answer 30

• contamination of the culture
• ## ensure safety to the performer

Question 31

a. [?] a clean microscopic slide
b. [?] a thin, uniform film of bacterial growth on a glass slide as describe depending on the source of the sample.
c. [?] on the glass slide over an area about 2-3 cm diameter to make a thin film.
d. [?] until the liquid evaporates.
e. [?] the air-dried smears.

Answer 31

a. Label
b. Aseptically transferring
c. Spread the specimen
d. Air-dry the smear
e. Heat-fix

Question 32

✓ If the smear is made correctly, it should look [?].
✓ If it is [?], there are too few bacteria, if it looks [?], there are too many bacteria. [?] results in too many cells thus microscopic examination of individual cells is not possible.

Answer 32

slightly milky (or slightly cloudy)
clear; milky; Thick smear

Question 33

✓ While the smear is [?], the cells are alive and should be treated as a biohazard.
✓ Do not [?] around or blow on it as this will disperse bacteria into the air.
✓ Do not use the [?] to dry the smear. Extreme heat cause cell distortion and splattering may occur.

Answer 33

wet
wave the slide
Bunsen burner

Question 34

process by which the microorganisms are killed while preserving the internal and external structures of cells. The microorganisms are preserved and attached in position on the microscopic slide.

Answer 34

Fixing microorganism

Question 35

heat kills the microbial cells by denaturing their protein, and the coagulated protein sticks the cells to the slide

Answer 35

Heat fixation

Question 36

use of chemical fixatives like ethanol, acetic acid, mercuric chloride, formaldehyde, and glutaraldehyde

Answer 36

Chemical fixation

Question 37

Heat fixation Technique:

i. Pass rapidly to an open flame [?] (for a second or two) without the dried film being exposed to the flame; or
ii. Use of slide warmer at

Answer 37

i. two or three times

ii. 60°C for at least 10 minutes

Question 38

Chemical fixation Technique:

A drop of the chemical agent is simply added to the liquid containing microorganisms. The fixative must preserve the morphology of host cells as well as bacteria. The method is especially useful for examining

Answer 38

bloody specimen material

Question 39

Chemical fixation Technique:

A drop of the chemical agent is simply added to the liquid containing microorganisms. The fixative must preserve the morphology of host cells as well as bacteria. The method is especially useful for examining

Answer 39

bloody specimen material

Question 40

Chemical fixation Advantage:

Answer 40

does less damage to the specimen than heat

Question 41

Chemical fixation Disadvantage:

Answer 41

distorts the cell’s appearance; motility cannot be studied

Question 42

Most bacteria when examined unstained have [?]. The purpose of preparing smears is to allow staining of the microorganisms. Once stained, microscopic examination allows examination of bacterial shape, size, and arrangements. There are two types of smears depending on the source of specimen:

Answer 42

no color

Question 43

Examination of Stained Specimens

Answer 43

1. Direct smear

2. Indirect smear

Question 44

preparation of the primary clinical sample (specimen collected from the patient) received in the laboratory for processing

Answer 44

Direct smear

Question 45

provides a mechanism to identify the number and types of cells present in the specimen, including white blood cells, epithelial cells, and predominant organism type

Answer 45

Direct smear

Question 46

Reasons for microorganisms not growing on culture but seen in the direct smear:

Answer 46

1. slow-growing organisms
2. a patient receiving antibiotic treatment preventing the growth of the microorganism
3. the microorganisms are no longer viable
4. the microorganisms require special media for growth
5. the specimen was not processed appropriately

Question 47

(the bacteria are present in the sample but did not yet grow on culture media)

Answer 47

slow-growing organisms

Question 48

(direct smear identifies nonliving cells which can no longer multiply thus any bacterial growth on culture)

Answer 48

a patient receiving antibiotic treatment preventing the growth of the microorganism

Question 49

(remember that there are growth factors that support the growth of fastidious bacteria)

Answer 49

the microorganisms require special media for growth

Question 50

Bacterial culture: propagation of bacteria on

Answer 50

artificial growth medium

Question 51

preparation where the primary sample has been processed in culture and the smear contains organisms following purification or growth on artificial media (solid or liquid)

Answer 51

Indirect smear

Question 52

ensure that the smear is not too thick

Answer 52

Indirect smear