Microbiological Lab Techniques

Question 1

Purpose of microscopy

Answer 1

To visualize smallmicroorganisms (diagnostic microbiology)

Question 2

Brightfield microscopy

Answer 2

Use of focused light to visualize small objects (objective x ocular lens = mag); example; wet mount

Question 3

Darkfield microscopy

Answer 3

Scattered or reflected light to visualize object; visualization of thin bacteria (spirochete)

Question 4

Fluorescent microscopy

Answer 4

One wavelength of light is used to cause another wavelength of light to be detected; fluorescent molecules target specific cell components

Question 5

Electron microscopy

Answer 5

Electrons used to visualize; high mag; transmission = electrons through; scanning = electrons bounce

Question 6

Purpose of direct examination

Answer 6

Visualize cells with minimal preparation

Question 7

Wet mount

Answer 7

Direct visualization in water/saline; no processing (i.e. vaginal wet mount)

Question 8

KOH

Answer 8

Usually 10% to dissolve proteinaceous non-fungal elements

Question 9

Lactophenol cotton blue

Answer 9

Type of KOH added to non-specifically stain material (ex. fungal elements)

Question 10

India ink

Answer 10

Type of KOH added to stain everything but yeast polysaccharide capsule (ex. cryptococcus)

Question 11

Iodine

Answer 11

Stain organelles darker than cytoplasm (ex. parasite cysts)

Question 12

Differential stain

Answer 12

Colored dyes that differentiate organisms or structures within organisms

Question 13

Acid fast stain

Answer 13

Stains mycobacterium, nocardia, cryptosporidium

Question 14

Fluorescent stains

Answer 14

Calcofluor white stain - interacts with cellulose and chitin in cell walls and fluoresces blue

Question 15

Wright-Giemsa stain

Answer 15

differential used to stain parasites, intracellular bacteria, viral elements, and eukaryotic cells (polychromatic stain that contains azure B, methylene blue, and eosin Y; eosin ions stain basic structures orange/pink, others stain acidic structures blue/purple

Question 16

Methenamine silver

Answer 16

Used to stain fungal elements and some bacteria in tissues

Question 17

Trichrome stain

Answer 17

Used to detect fecal protozoa

Question 18

Gram stain

Answer 18

1.) Heat fix
2.) Crystal violet solution - purple stain is positively charged; enters bacterial cell and complexes with anionic molecules
3.) Gram’s iodine - iodine acts as a mordant; interacts with crystal violet and forms a larger complex
4.) Gram’s decolorizer - alcohol dehydrates; peptidoglycan wall shrinks and captures crystal-violet iodine complex within cell
Gram + = thick cell wall doesn’t let crystal violet through
Gram - = thin cell wall allows stain through
5.) Safranin - red counter stain that stains whatever primary stain didn’t (Gram - )

Question 19

Ziehl-Neelsen and Kinyoun stains

Answer 19

Same principle as gram stain but for mycobacterium; carbofushin (purple dye) added; alcohol washes what didn’t stain; methylene blue (green) acts as counter stain

Question 20

Auramine-rhodamine

Answer 20

Fluorescent stain of mycolic acids

Question 21

Purpose of culture medium

Answer 21

• select for growth of specific microorganisms

- differentiate amongst microorganisms

Question 22

Nonselective culture

Answer 22

Supports growth of many organisms

Question 23

Trypticase soy/broth agar

Answer 23

Digested soybean meal and casein; full of nutrients (supports growth of many organisms)

Question 24

Nutrient broth/agar

Answer 24

Peptone and beef extracts; full of nutrients

Question 25

Blood agar

Answer 25

Trypticase soy with sheep blood (enriched)

Question 26

Chocolate agar

Answer 26

Trypticase soy with lysed sheep blood (enriched)

Question 27

Thioglycolate broth

Answer 27

Sodium thioglycolate added to promote a reducing (anaerobic) environment; used to grow anaerobic bacteria and can be used to differentiate oxygen usage

Question 28

Sabouraud dextrose agar

Answer 28

Growth of fungi

Question 29

Selective culture; differential

Answer 29

Supports growth of specific organisms usually by limiting growth of others; differentiation of organisms in a mixture

Question 30

MacConkey agar

Answer 30

Selective for gram- ; differential for lactose fermentation

Question 31

Mannitol salt agar

Answer 31

Selective for gram+ Staphylococci; differential for S aureus (mannitol fermentation)

Question 32

Lowenstein-Jensen agar

Answer 32

selective for mycobacteria species

Question 33

Eosin-methylene blue agar

Answer 33

Selective for gram- ; differential for lactose fermentation

Question 34

Hektoen enteric agar

Answer 34

Selective for gram - ;differential for hydrogen sulfide production (black) and lactose/sucrose/salicin formation (pink)

Question 35

Specialized culture

Answer 35

Ingredients support the growth of specific organisms

Question 36

Thayer martin

Answer 36

Neisseria gonorrhoeae

Question 37

Buffered charcoal yeast extract (BCYE) agar

Answer 37

Legionella

Question 38

Thiosulfate citrate bile salts sucrose (TCBS) agar

Answer 38

Vibrio species

Question 39

Regan-Lowe agar and Bordet-Gengou agar

Answer 39

Bordetella pertussis

Question 40

Purpose of biochemistry in lab techniques

Answer 40

Differentiation of bacteria based on varying biochemical pathways

Question 41

Catalase test

Answer 41

Bubbles = + catalase; catalase enzyme functions as part of the antioxidant defense response; breaks down H2O2 to O2 and 2H2O

Question 42

Oxidase test

Answer 42

Purple = positive (before 30 secs); some bac use O2 as final electron acceptor (reduce O2 to form H2O via cytochrome oxidase complex)

Question 43

Antibiotic sensitivity test (Kirby-Bauer, Disk Diffusion)

Answer 43

Wells with increasing amounts of antibiotics are inoculated with clinical sample; resistant = will grow, sensitive = no growth; first well in an antibiotic series with no growth is the minimum inhibitory concentration

Question 44

Purpose of immunology in labs

Answer 44

Use of immune components (usually antibodies) to differentiate microorganisms

Question 45

Examples of immunology in lab

Answer 45

Latex agglutination, immunofluorescence, ELISA, western blot, and others

Question 46

Latex agglutination

Answer 46

- Latex bead solution (with antibody coating) added to flat surface - mix bacterial strain in - evaluate for precipitation (precipitate = +)

Question 47

Immunofluorescence

Answer 47

Use of antibodies that are conjugated to fluorescent molecules

Question 48

Purpose of eukaryotic cell culture

Answer 48

Grow intracellular bacteria and viruses; eukaryotic cells act as the culture medium for intracellular bac and viruses