Microbiology Techniques

Question 1

Recognize the varied purposes for culture media used in the clinical microbiology laboratory.

Answer 1

Promote survival, growth, and/or reproduction of microorganisms

 Increase antigen production
 Allow for selective growth
 Identification of various biochemical end products
 Other biological characteristics (pigment, spores, hemolysis, etc.)

Question 2

**Define the following terms as they apply to media and list examples of each, as discussed in class: defined

** quiz question

Answer 2

Defined
 Ideal for all media
 Known amounts of pure chemical substances
 Predictable and Reproducible
 i.e.

Question 3

**Define the following terms as they apply to media and list examples of each, as discussed in class: primary isolation (nonselective)

** quiz question

Answer 3

Primary Isolation
 Referred to as “non-selective” or “nutrient media”
 Supports a large number of organisms
 i.e. Trypticase Soy, Sheep Blood Agar

Question 4

**Define the following terms as they apply to media and list examples of each, as discussed in class: selective

** quiz question

Answer 4

Selective Isolation
 Contains inhibitors to hinder the growth of some organisms
 i.e. CAN

Question 5

**Define the following terms as they apply to media and list examples of each, as discussed in class: differential

** quiz question

Answer 5

Differential
 Indicators allow recognition of certain biochemical reactions
 i.e. MAC

Question 6

**Define the following terms as they apply to media and list examples of each, as discussed in class: enrichment

** quiz question

Answer 6

Enrichment
 Promote growth of some organisms
 Used for the isolation of fastidious organisms
 Often incubated in C02 incubator
 i.e. CHOC

Question 7

**Define the following terms as they apply to media and list examples of each, as discussed in class: maintenance

** quiz question

Answer 7

Maintenance
 Allows survival of organisms for extended period of time
 i.e.

Question 8

Match different selective agents with the type of organism (GPC, GNR, etc.) inhibited by the agent.

Dyes?

Answer 8

 Dyes – Brilliant Green, Eosin, Methylene Blue, Basic Fuchsin – mainly inhibits GP organisms

Question 9

Match different selective agents with the type of organism (GPC, GNR, etc.) inhibited by the agent.

Heavy Metals?
Antimicrobials?

Answer 9

 Heavy Metals – Bismuth: inhibits GP and most GN organisms (exception: Salmonella)
Antimicrobials – no further details are needed at this point

Question 10

Match different selective agents with the type of organism (GPC, GNR, etc.) inhibited by the agent.

Other Chemicals?

Answer 10

Other Chemicals
 Citrate: inhibits GP
 Sodium deoxycholate: inhibits GP
 Phenylethyl alcohol: inhibits GN
 Azide: inhibits GN
 NaCl: inhibitory to most organisms except Staphylococcus

Question 11

Describe how the choice of media is made for the setup of different specimens.

Answer 11

Type of clinical specimen (sterile vs. usual flora)
 Always include primary isolation media
 Areas with flora usually require use of selective and differential media
 Enrichment media often added on specimens from normally sterile areas

 Nature of clinical condition
 Results of preliminary exam of specimen

Question 12

Interpret hemolytic reactions on SBA.

Answer 12

 Gamma – the organism does not possess hemolytic enzyme; therefore, no destruction of RBCs
 Beta – complete lysis of the RBCs which leads to a clearing of the auger plate where the organism is
 Alpha – partial lysis of the RBCs, appears darken with a green-gray color of the auger plate where the organism
is

Question 13

**Identify the purpose and type of medium (differential, selective, etc.) for each of the following: (Example: MAC is selective for GNRs and differential for lactose fermentation)

SBA (SAP, BAP)

** quiz question

Answer 13

SBA (SAP, BAP) Primary (non-selective) media – best medium for colony morphology, used to determine hemolysis

Question 14

**Identify the purpose and type of medium (differential, selective, etc.) for each of the following: (Example: MAC is selective for GNRs and differential for lactose fermentation)

CNA

** quiz question

Answer 14

Colistin Nalidixic Acid Agar (CNA) Selective for GP

Question 15

**Identify the purpose and type of medium (differential, selective, etc.) for each of the following:

MAC

** quiz question

Answer 15

MacConkey Agar (MAC) Selective for GNRs and differential for lactose fermentation

Question 16

**Identify the purpose and type of medium (differential, selective, etc.) for each of the following: (Example: MAC is selective for GNRs and differential for lactose fermentation)

Chocolate (CHOC)

** quiz question

Answer 16

Chocolate Agar (CHOC) Enrichment – SBA heated to lyse RBCs to provide hemoglobin and other enrichments

Question 17

**Identify the purpose and type of medium (differential, selective, etc.) for each of the following: (Example: MAC is selective for GNRs and differential for lactose fermentation)

XLD

** quiz question

Answer 17

Xylose, Lysine, Deoxycholate (XLD) Selective for GNRs and differential for lactose fermentation and other carbohydrate use

Question 18

**Identify the purpose and type of medium (differential, selective, etc.) for each of the following: (Example: MAC is selective for GNRs and differential for lactose fermentation)

HEK

** quiz question

Answer 18

Hektoen Agar (HEK) Selective for GNRs and differential for lactose fermentation and “other other” (diff. from XLD) carbohydrates

Question 19

**Identify the purpose and type of medium (differential, selective, etc.) for each of the following: (Example: MAC is selective for GNRs and differential for lactose fermentation)

EMB

** quiz question

Answer 19

Eosin Methylene Blue Agar (EMB) Selective for GNRs and differential for a “green metallic sheen” (i.e. E. Coli)

Question 20

**Identify the purpose and type of medium (differential, selective, etc.) for each of the following: (Example: MAC is selective for GNRs and differential for lactose fermentation)

Bismuth Sulfite

** quiz question

Answer 20

Bismuth Sulfite Selective for Salmonella

Question 21

**Identify the purpose and type of medium (differential, selective, etc.) for each of the following: (Example: MAC is selective for GNRs and differential for lactose fermentation)

Thioglycollate (Thio)

** quiz question

Answer 21

Thioglycollate (Thio) Primary (non-selective) media – frequently used liquid medium for cultivation of organisms from original specimens (i.e. swabs, CSF, etc.)

Question 22

**Identify the purpose and type of medium (differential, selective, etc.) for each of the following: (Example: MAC is selective for GNRs and differential for lactose fermentation)

Brain Heart Infusion (BHI)

** quiz question

Answer 22

Brain Heart Infusion (BHI) Primary (non-selective) media

Question 23

**Identify the purpose and type of medium (differential, selective, etc.) for each of the following: (Example: MAC is selective for GNRs and differential for lactose fermentation)

Martin Lewis/Thayer Martin

** quiz question

Answer 23

Martin Lewis/Thayer Martin Enrichment and selective for non-normal flora

Question 24

Compare and contrast the purposes of Chocolate and Martin Lewis (or Thayer Martin) agar.

Answer 24

Both media utilize a chocolate agar, an enrichment medium, that supports growth of most organisms, specifically fastidious organisms – however, the chocolate agar does not contain any selective agents, as ML/TM contains antimicrobials that will inhibit growth of normal flora

Question 25

Identify the most commonly used, effective disinfectant for non-metallic lab surfaces.

Answer 25

Sodium Hypochlorite (Bleach)

Question 26

List the standard temperature, psi and time used in autoclaving most lab materials.

Answer 26

121 degrees Celsius, 15 psi, 15-20 minutes

Question 27

Categorize the following as aerobic or anaerobic organisms: obligate aerobe, microaerophile, facultative anaerobe, aerotolerant anaerobe, moderate obligate anaerobe and strict obligate anaerobe.

Answer 27

Aerobic organisms (able to utilize O2 for growth) Anaerobic organisms (unable to utilize O2 for growth)
Obligate aerobe Aerotolerant anaerobe (can survive in the presence of O2)
Microaerophile Moderate obligate anaerobe
Facultative anaerobe Strict obligate anaerobe (cannot survive in the presence of O2)

Question 28

State the category of aerobes which is most commonly encountered in the clinical laboratory.

Answer 28

Facultative anaerobe – can perform aerobic respiration and anaerobic fermentation depending on their environment, but prefer to use O2

Question 29

**Differentiate between gas concentrations in ambient (non CO2), CO2 (partial O2), microaerophilic and anaerobic incubation conditions.

** quiz question

Answer 29

Ambient Air (non-CO2): 21% O2, 0.03% CO2 (room air)
CO2 Incubator: 15% O2, 5-10% CO2
Microaerophilic: 5% O2, 10% CO2
Anaerobic: 0% O2, 5-10% CO2

Question 30

**Choose the best incubation condition for primary plates, biochemical tests, antimicrobial susceptibility tests, differential media, fastidious organisms, and anaerobic organisms.

** quiz question

Answer 30

Ambient Air CO2 Incubation (capnophilic)
Biochemicals Primary plates
Differential media Fastidious organisms (i.e. Chocolate agar – enrichment media)
Antimicrobial susceptibility tests Anaerobic organisms

*sensitivity to pH changes, in the presence of CO2, is why biochemicals & antimicrobial susceptibility prefer ambient air

Question 31

Discuss the advantages of performing a direct smear.

Answer 31

A direct smear is a GS done directly on a clinical specimen

 Provide initial clue to cause of infection
 Evaluate quality of specimen
 Need for additional media?
 QC for culture results

Question 32

**Outline the steps used and the purpose for each step in the Gram stain – discuss the preparation of a smear

**quiz question

Answer 32

Preparation of smear

 From a colony (solid growth on agar plate)
 Add drop of sterile water or saline to slide
 Emulsify small amount of a single colony

 From a broth or direct smear
 No water/saline
 Add 1 drop of specimen/broth directly to slide
 Spread drop with sterilized loop for even drying

 Air dry smears
 Heat fixation vs. Methanol fixations (preferred method due to maintain cell morphology)

Question 33

**Outline the steps used and the purpose for each step in the Gram stain.

** quiz question

Answer 33

Stain Procedure
 Crystal Violet – primary stain
 Add 2-3 drops of sodium bicarbonate buffer, in certain situations, to prevent the crystallization of the Crystal
Violet [15 sec. – 1 min.] – rinse with H2O
 Iodine – mordant [15 sec. – 1 min.] – rinse with H2O
 Complexes with Crystal Violet within the cells and prevents it from washing out
 Acetone/Alcohol – decolorizer (differential step) [no longer than 5 sec – “until runoff is clear”] – rinse with H2O
 Safranin – counterstain [let slide stand for 1 minute] – rinse with H2O
 Will only pick-up the counterstain if the Crystal Violet was washed away – if Crystal Violet remains, it will not
pick-up the counterstain
Observation – usually 100x oil immersion

Question 34

**Outline the steps used and the purpose for each step in the Gram stain, SPECIFICALLY, include a discussion of the cell wall composition of Gram negative and Gram positive organisms.

** quiz question

Answer 34

Gram Positive (dark blue-purple) – increased peptidoglycan allowing the crystal violet-iodine complex to become bound and to prevent being washed away by the decolorizer

Gram Negative (pink-red) – increased lipids – crystal violet-iodine complex will attach to the peptidoglycan within the lipids, but the decolorizer will disintegrate the complex out of the lipids and it will wash away, the counterstain will be picked up by the lipids

Question 35

**Describe the appearance of a correctly Gram stained buccal smear.

** quiz question

Answer 35

A squamous cell that possesses a pink nucleus and cytoplasm indicating Gram Negative – Gram variable bacteria should be present

Question 36

**Troubleshoot problems that are commonly seen in the Gram stain procedure. (e.g. overdecolorization, etc.)

During the GS, I seemingly had a GN organism, but forgot to do the decolorizing step?
During the GS, I seemingly has a GP organism, but forgot the iodine?
Undercolorization?
Overcolorization?

** quiz question

Answer 36

 During the GS, I seemingly had a GN organism, but forgot to do the decolorizing step?
 It would falsely be interpreted as a GP organism (decolorizer is required to wash away CV-Iodine complex)
 During the GS, I seemingly has a GP organism, but forgot the iodine
 It would be falsely interpreted as a GN organism (would not form CV-Iodine complex)
 Undercolorization – leading to GN retaining some of the CV-Iodine complex (GN displays as GP)
 Overcolorization – leading to ONLY GN being observed because the CV-Iodine complex was rigorously washed
away (GP displays as GN)

Question 37

Identify the purpose(s) for using each of the stains discussed in lecture

Answer 37

 Acridine Orange- fluorescent stain for hard to visualize bacteria: stains all nucleic acids
 Ziehl Neelsen or Kinyoun – AF stains: interpretation – red (positive), blue (negative)
 Calcofluor White – fluorescent fungal stain

Question 38

Identify microorganism characteristics as either phenotypic or genotypic

Answer 38

 Genotypic
 Organism’s genetic makeup (genes and nucleic acids)
 Phenotypic
 Observable characteristics/features
o Microscopy/Macroscopy morphology
o Staining
o Environmental and nutritional requirements, etc.

Question 39

What does “MALDI-TOF” stand for?

Answer 39

“Matrix-assisted Laser Desorption Ionization - Time of Flight”

Question 40

Recognize the importance of doing quality control on media, regardless of in-house preparation or commercial purchase.

Answer 40

 Each lot must be visually examined and tested for:
 Sterility
 Support of appropriate growth
 Appropriate biochemical reactions