Microbiology 1 Lab Practical 1

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1

MPN

(Most Probable Number) indirect/viable method, live cells. Make statistical estimates numbers of cells by their patterns of growth in liq. culture media

2

Standard plate count (SPC)

Direct/viable, live cells, good for patients. Dilute a sample in saline, spread on solid media & count colonies. Calculate the # of cells in original sample from counts & dilution. *most common*

3

Spectroscopy

Indirect/total live & dead, not good for patients. Cells dead & alive. Measure the amount of light that passes thru a liq. culture using a spectrophotometer & est. the # of cells/mL based on amount of light that passes through the culture. * most common *

4

DAPI or AODC staining & microscopy

Direct/total. Live/dead. Stain the cells w/fluorescent dyes, which make them visible in raw samples (ie soil). Count the # of cells using a fluorescent microscope.

5

# of colonies needed on media to give accurate counting results

30-300

6

Spectroscopy method of counting (how it works)

based on turbidity. Optical density used to describe the turbidity of a culture. The Spec 20 can quantify the OD of a culture indirectly by measuring the amount of light that can pass through a a culture (percent transmittance %T). The higher the OD the lower the %T (indirectly proportional, the higher the OD the higher the turbidity (directly proportional)

7

If you were given a stock culture that was really old, would you expect the SPC to be higher or lower?

Lower because most bacteria in death phase

8

With SPC/dilution, why bother doing 3 replicates?

To normalize & standardie counts

9

Why shouldn't you use a plate that has 10 colonies to calulate the number of cells in the starting culture?

Not enough to get statisical validity. NETC

10

Which counting method would you use if you need isolated colonies from a soil sample and why?

DAPI or AODC staining. You can apply flourescent stain to bacteria you want to to see and use fluoresent microsope.

11

Kirby-Bauer Technique

Using paper disks impregnated w/different antibacterial medicines on Mueller-Hinton agar plate to determine the sensitivity of a bacterium to serveral antibacterial medicines. No growth or halo around disk = sensitive (zone of inhabition--use whole diameter). Grows right up to the disk = resistant.

12

Robert Kock

In the 1880s he developed streaking for isolation.

13

Aseptic technique

Lab technique used to exclude contaminants.

14

Why is it desirable that microscope objectives be parfocal?

So you can focus it once, then switch objectives and refocus only using fine adjustment knob.

15

Which objective focuses closest to the slide?

100x Oil immersion

16

What controls the amount of light reaching the oculars?

Iris diaphragm (opens/closes)and condensor (up/down)

17

What effect does increased magnification have on the field of vision?

Raise magnification lowers field of view

18

What does the "ubiquity of microorganisms" refer to ?

The concept that microorganisms are everywhere

19

Fastidious organisms

Live in body and have "finicky" nutrient requirements

20

What are the objective powers and names?

4x scanning, 10x low power, 40x high powerh(high dry), 100x oil immersion

21

Primary purpose of: deeps, slants, broths?

Deeps= ovservie O2 requirements and motility. Slants = surface growth and pattern. Broths = large numbers of bacterial growth.

22

Resolution (or resolving power)

The ability of lenses to reveal fine detail or 2 points distinctly separated.

23

When using low-power lense, the iris diaphragm should be...?

Barely open so that good contrast is achieved. More light is needed w/higher magnification.

24

Darkfield microscopy is valuable for observing...?

Bacterium that are not stainable with conventional stains, but can be observed in directd smears (ex. Treponema pallidum - syphilis).

25

Brownian movement

Not true motility but rather the movement caused by molecules in the liquid striking an object. Vibrates or quivers.

26

Shape of most bacteria grown in the lab

Cocci or rods

27

What is the usual % agar and what is a lower % used for?

1.5% agar is normal. Semisolid deeps 0.5-o.7% agar can be used to determine motility.

28

Slants vs. plates

Slants also provide a solid growth surface, but are easier to store and transport.

29
card image

The 6 patterns of growth on agar slants

card image

Arborescent (branched-looks like tree
Beaded
Echinulate (pointed)
Filiform (even)
Rhizoid (rootlike)
Spreading

30

CFU

Colony forming unit

31

Where is isolation of bacteria frequently used?

On selective media

32

Selective Media

Designed to encourage growth of certain types of organisms

33

Differential Media

Contain indicators that expose differences between organisms (i.e. pH)

34

Enteric

Gut bacteria, usually Enterobacteriaceae. Coliforms are enteros & lactosefermentators, noncoliform do not ferment lactose

35

Bacteroides fragilis

The most abudnand bacteria in the human colon. 10^11 cells per gram of feces. Also the most common anerobic pathogen. Used in BBE AGar

36

Growth patterns in agar & broth

-Colony shape--circular, irregular, punctiform (tiny)
-Margin--entire(smooth, undulate, lobated, filamentous, rhizoid (branched root)
-Elevation--flat, raised, convex, pulvincate (very convex_, umbonate (raised in center)
-Texture--moist, dry, mucoid
-Pigment--opaque, translucent, shiny, dull
-BROTH--pellicles (dots), sediment (at bottom), uniform fine turbidity, flocculent (clump)

37

Aerotolerance determined in what media?

Agar deep stabs and shakes

38

Obligate aerobes

Require O2, grow at top

39

Facultatvie anearobes

Grow with or without O2, grow throughout media but more dense on top

40

Aerotolerant anaerobes

Don't require O2, but not aversely affected by it. Uniform growth throughout media

41

Microaerophiles

Survive only in lower than atmospheric levels of O2, middle or upper middle growth in media.

42

Capnophiles

Are microaerophiles that must have elevated CO2

43

What type of agar used in agar deep stabs?

Tryptic soy agar

44

Bile salts

Used in selective media, selects against organisms incapable of surviving gut (especially gram +)

45

Lactose

Helps distinguish between fermtors and nonfermentors

46

Thiosulfate

Used to id organisms cable of reducing sulfure to H2S

47

Ferric Iron

Inicator of sulfur reducers, black precip


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