Microbiological Methods-Steinauer

Question 1

What are the 3 types of specimen collection?

Answer 1

Direct, Indirect, and Site with normal flora.

Question 2

What specimen collection type is described as: The pathogen is localized in an otherwise sterile site, and a barrier such as the skin must be passed to sample it. This may be done surgically or by needle aspiration. The specimen collected contains only the pathogen.
Provide 2 examples.

Answer 2

Direct; Examples: deep abscess and cerebrospinal fluid.

Question 3

What specimen collection type is described as: The pathogen is localized, but must pass through a site containing normal flora in order to be collected (non-sterile site). The specimen contains the pathogen, but is contaminated with the nonpathogenic flora. The degree of contamination is often related to the skill with which the normal floral site was “bypassed” in specimen collection. Provide 2 examples.

Answer 3

Indirect; Examples: expectorated sputum and voided urine.

Question 4

What specimen collection type is described as: The pathogen and nonpathogenic flora are mixed at the site of infection. Both are collected and the non-pathogen is either inhibited by the use of selective culture methods or discounted in interpretation of culture results.
Provide 2 examples.

Answer 4

Site with normal flora; Examples: throat and stool

Question 5

What type of microscopy is described as: Bright background, typically use a stain to see the microbes. Resolution: (0.2μm).

Answer 5

Bright field

Question 6

What type of microscopy is described as: Block out indirect light, background is dark, specimen is light. Only picks up the light scattered by the specimen. Resolution: 0.15 to 0.2 μm.

Answer 6

Dark field

Question 7

What type of microscopy is described as: fluorophore (tag) attached to specific molecule for diagnosis.

Answer 7

Fluorescence

Question 8

What can you learn from microscopy?

Answer 8

1. Size, shape and morphology.
2. Simple stains: stain all bacteria one color.
3. Can use differential stains to stain specific bacteria.

Question 9

What are the steps (4) of gram staining? What color are gram +/gram - bacteria? What does the thick peptidoglycan layer of gram + bacteria do?

Answer 9

1. Crystal violet added to specimen (purple stain).
2. Iodine to set the stain.
3. Alcohol wash to destain.
4. Then add the counterstain, safranin (red stain).
   Gram+ = purple
   Gram- = red
   The thick peptidoglycan layer of gram+ bacteria functions to retain the dark purple crystal violet stain.

Question 10

What organisms (6) CANNOT be gram stained? Why?

Answer 10

1. Mycobacteria (TB)- too much lipid in cell wall
2. Treponema pallidum (Syphilis)- too small
3. Mycoplasma pneumoniae (Walking pneumonia)- no cell wall
4. Legionella pneumophila (-) (Legionnaire’s Disease)- poor uptake of safranin
5. Chlamydia (-)- intracellular, very small
6. Rickettsia (-) (tick and other insect borne fevers)-intracellular, very small

Question 11

What is culturing? What are the phases (4) of bacterial growth (in a closed system)?

Answer 11

Growing or multiplying organisms from a sample of interest by letting them reproduce. (either on plate or broth)
Phases of bacterial growth (closed system):
1. Lag phase (metabolic activity but w/o division, getting ready)
2. Exponential phase (dividing stage)
3. Stationary phase (growth slowed, nutrients depleted, spores formed)
4. Death phase (no more nutrients, toxic wastes build up)

Question 12

Why is quantification important? What is the main method of quantification?

Answer 12

Quantifying helps to determine if it is a true infection or just contamination.
Main Method: Serial dilution (1mL of original inoculum : 9 mL broth)-Determine the colony forming units (CFU) by multiplying the number of countable colonies by the dilution factor.

Question 13

What is culture media? What are the environmental conditions (3) to be considered? What are the 4 atmospheric requirements to consider?

Answer 13

Sterile, “Soup” that contains elements to meet metabolic requirements of organism
•Liquid broth or solid gel: agar
•Environmental Conditions: 
1. Temperature 
2. pH
3. Atmospheric requirements:
-Obligate anaerobes: no oxygen
-Facultative: grow under both conditions
-Obligate aerobes: require oxygen
-Capnophilic: need increase CO2 tension

Question 14

What are the 3 basic types of culture media? What do they contain? What are they used for?

Answer 14

1. Nutrient-Contain growth requirements
2. Selective-Selects specific organisms (antimicrobials, bile salts, dyes, etc.). Useful when contaminants present.
3. Indicator or Differential-Identification. Contain substances that demonstrate a specific characteristic of a suspected pathogen.

Question 15

What type of media utilizes hemolysis as an indicator? What are the 3 possible results of this media?

Answer 15

Indicator or Differential.

1. Beta: complete hemolysis; clearing around the colony due to enzyme streptolysin
2. Alpha: incomplete hemolysis.
3. Gamma: no hemolysis at all.

Question 16

Is MacConkey agar selective or differential? What is it used for?

Answer 16

BOTH!
1. Differentiate lactose from non-lactose fermenting bacteria thru color change. (ferment lactose → red color)
2. Selective based on bile salts (enteric bacteria are resistant to bile salts, Gram+ cannot grow).
So it differentiates between lactose and non-lactose fermenting gram negative rods.

Question 17

What are the 5 tests for identification?

Answer 17

1. Sensitivity test (disk diffusion)
2. Coagulase test
3. Catalase test
4. Oxidase test
5. Immunologic tests.

Question 18

What type of ID test is described as: Take specimen, put on plate and add a disc soaked in an antibiotic. Incubate plate and determine the level of sensitivity based on how much clearing there is.

Answer 18

Sensitivity test (disk diffusion).

Question 19

What type of ID test is described as: Determines production of enzyme coagulase.Coagulase crosslinks the α and β chain of fibrinogen in plasma to form fibrin clot (clotting test). What does a positive test look like? Is it a rapid test? What is it used to ID specifically?

Answer 19

Coagulase test.
Positive test = clots
rapid test
Used to ID Staphylococcus aureus.

Question 20

What type of ID test is described as: Determines presence of catalase. Hydrolyzes hydrogen peroxide to water and oxygen. What does a positive test look like? Is it a rapid test? What is it used to ID specifically?

Answer 20

Catalase Test.
Catalase positive = bubbles form.
rapid test
2H2O2 → 2H20 + O2
Differentiates Staphylococcus (+) from Streptococcus (-).

Question 21

What type of ID test is described as: Determines if bacterium produces certain types of cytochrome c oxidases. What does a positive test look like? Is it a rapid test? What is it used to ID specifically?

Answer 21

Oxidase test. OX+ or OX-. Redox indicator that changes color when oxidized.
rapid test
Positive test = color changes to purple.
This test only tests a specific enzyme. This does not mean that a result of oxidase negative would mean the bacterium is anaerobic.

Question 22

What type of ID test is described as: Based on specific binding of antibody and antigen. Use in immunofluorescence: Either direct or indirect. Sample is bound to a glass microscope slide. A specific antibody for an antigen of the pathogen of interest is then added to the specimen. This antibody is labeled w/ a fluorescent tag. Slide is then washed to remove non-bound antibodies.

Answer 22

Immunologic tests.

Question 23

What is ELISA? Describe the 2 types.

Answer 23

Enzyme-linked immunosorbent assay. Color change.
1. Antibody ELISA:
oDetect antibodies in patient tissue.
oStart w/ a specific antigen in the wells.
2. Antigen ELISA
oDetect antigen in patient tissue.
oAn antibody specific to the antigen is bound to the wells of a plate. Then add sample. Next an enzyme linked antibody specific to the first antibody is added. This antibody binds. When a substrate specific to the enzyme is added, the enzymatic reaction causes a color change which can be measured. Positive test = color change.

Question 24

Describe the rapid diagnostic test mechanism.

Answer 24

A swab containing the sample is inoculated onto a nitrocellulose membrane which contains in serial order, antibodies specific to the antigen with a conjugated tag for visualization, a line of specific antibodies to the antigen (test line), and a control line which contains antibodies specific to the conjugated antibodies. Capillary flow drives the specimen across the membrane and if the antigen is present, it first binds to the antibodies with the conjugated tag. Next, this complex binds with the antibodies on the test line, and then the antibodies on the control line. Thus, a positive sample results in visual lines at both the test line and the control line. A negative sample results in a visual line only at the control line.

Question 25

Define sensitivity.

Answer 25

oProportion of actual positives which are correctly identified as such
oPercentage of sick people actually identified as such by the test

Question 26

Define specificity.

Answer 26

oProportion of actual negatives which are correctly identified as such
oThe percentage of healthy people who are correctly identified as not having the infection.