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

MPN

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*

Standard plate count (SPC)

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 *

Spectroscopy

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.

DAPI or AODC staining & microscopy

30-300

# of colonies needed on media to give accurate counting results

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)

Spectroscopy method of counting (how it works)

Lower because most bacteria in death phase

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

To normalize & standardie counts

With SPC/dilution, why bother doing 3 replicates?

Not enough to get statisical validity. NETC

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

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

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

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.

Kirby-Bauer Technique

In the 1880s he developed streaking for isolation.

Robert Kock

Lab technique used to exclude contaminants.

Aseptic technique

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

Why is it desirable that microscope objectives be parfocal?

100x Oil immersion

Which objective focuses closest to the slide?

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

What controls the amount of light reaching the oculars?

Raise magnification lowers field of view

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

The concept that microorganisms are everywhere

What does the "ubiquity of microorganisms" refer to ?

Live in body and have "finicky" nutrient requirements

Fastidious organisms

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

What are the objective powers and names?

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

Primary purpose of: deeps, slants, broths?

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

Resolution (or resolving power)

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

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

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

Darkfield microscopy is valuable for observing...?

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

Brownian movement

Cocci or rods

Shape of most bacteria grown in the lab

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

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

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

Slants vs. plates

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

The 6 patterns of growth on agar slants

Colony forming unit

CFU

On selective media

Where is isolation of bacteria frequently used?

Designed to encourage growth of certain types of organisms

Selective Media

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

Differential Media

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

Enteric

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

Bacteroides fragilis

-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)

Growth patterns in agar & broth

Agar deep stabs and shakes

Aerotolerance determined in what media?

Require O2, grow at top

Obligate aerobes

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

Facultatvie anearobes

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

Aerotolerant anaerobes

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

Microaerophiles

Are microaerophiles that must have elevated CO2

Capnophiles

Tryptic soy agar

What type of agar used in agar deep stabs?

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

Bile salts

Helps distinguish between fermtors and nonfermentors

Lactose

Used to id organisms cable of reducing sulfure to H2S

Thiosulfate

Inicator of sulfur reducers, black precip

Ferric Iron