MICROM 402 Final

purple nonsulfur bacteria

gram -, store sulfur outside of the cell(no granules), fix nitrogen from air to urea/ammonia

anaerobic phototroph

use light as energy source in the absence of oxygen

enrichment culture

a culture medium used for preliminary isolation that favors the growth of a particular microorganism

goal of aseptic technique

prevent microbial contamination

pure culture

contains only one species or strain

single colony isolation

technique to get a pure inoculum or culture

empty magnification

Magnification without an increase in resolution.

refraction

The bending of a wave as it passes at an angle from one medium to another

simple stain

a method of staining microorganisms with a single basic dye

differential stain

a procedure that stains specific morphological structures - usually a multiple stain

specialized stains

detect specific structures or specific cell types such as flagella or endospores. Detection of PHB bodies

what does iodine do in gram stain

binds to the primary stain and prevents the stain from leaving the cells with a thick peptidoglycan cell wall

Decolorization

Is the step that occurs between the application of the 2 basic stains. crystal violet and safranin. washes out crystal violet color of gram negative cells

methylene blue stain

Stains organic tissue. Needed to see animal cells underneath microscope.

endospore stain

This is a differential stain used to detect the presence and location of spores in bacterial cells.

Endospore stain procedure

1) Prepare smear with Bacillus Culture, same technique as simple stain procedure.

2) Air dry and heat fix smear and place on staining bars

3) Cover smear with Malachite Green

4) Gently heat the slide with stain with steam over boiling water

5) Steam slide for 5 minutes, replenish malachite green as it begins to evaporate. cover ith paper towel and saturate with more malachite green

6) Pour excess malachite green from slide and allow to cool, once slide is cool rinse with water and get all green off slide

7) Cover smear with Safranin for 1 minute

8) Pour off excess Safranin and use water bottle to rinse any left over safranin from slide

9) Gently blot slide using bibulous paper, then observe slide with immersion oil

Azotobacteria

- Gram-negative to Gram-variable

- nitrogen fixers!

- much larger than most bacteria

- commonly contain poly-(beta)-hydroxybutyrate (PHB)

- obligately aerobic chemoheterotrophs

- makes a slime layer to help create anaerobic respiration happen (makes a barrier to oxygen)

Azomonas

- do not form cysts

- isolated from freshwater habitats

- urease-negative, making them capable of utilizing ammonia but not urea as a nitrogen source

Azotobacter

free living nitrogen fixing bacteria found in soil. urease positive

enrichment for nitrogen fixing bacteria

inoculating soil or water samples into nitrogen free broth and shaking the culture to aerate it fully

- ashbys glucose broth, agar and ashbys butanol agar

- urea agar

Urease Test

Urease positive = PINK

Only E. colis and E. aerogenes are NEGATIVE (no color change, yellow)

Tests for urease activity as a source of reduced nitrogen

anoxygenic phototrophs

use hydrogen sulfide or organic compounds as a source of electrons.

purple nonsulfur bacteria enrichment

ATY broth incubated at room temperature with constant illumination by placing it by 40W lamp. ATY agar in anaerobic chambers

Gram positive cocci

Staphylococcus

Streptococcus

Enterococcus

micrococcus

type genus of the family Micrococcaceae, obligate aerobes, catalase positive, gram + cocci found in pairs of clusters, nonpathogenic, grows well at 25*C, cant tolerate high salt

Staphlococcus

cluster of cocci, falcultative anaerobes, catalase positive

obligate aerobic bacteria

gather at the top of the test tube in order to absorb maximal amount of oxygen

facultative anaerobes

can survive with or without oxygen

What happens when staph is grown on glycerol

they produce acids

where does micrococcus grow

drier regions

where does staphylococcus grow

moister, oilier regions

selective media

suppress unwanted microbes and encourage desired microbes

Mannitol Salt Agar

selective and differential, selective for salt tolerance, staph

Isolation of Micrococcus

TSY agar

Glucose ferment tube staph

ability to ferment glucose: staph should ferment glucose without gas production. Pink with no air bubble in durham vial

Mannitol fermentation test

pink is positive result, yellow means doesnt ferment mannitol

Agar Shake Tube

colonies form embedded in agar (works well for anaerobes)

shake tube: growth on top part

Aerobic: maximum O2 concentration - use for O2 for energy harvesting purpose

oxidase positive

aerobe

shake tube growth: growth throughout with more at surface

facultative anaerobe

catalase negative

anaerobe

shake tube growth: only at the bottom of tube

obligate anaerobe

acinetobacter vs psuedomonads

use oxidase test

yeast-glycerol + bromserol purple plate

solid line of growth with yellow halo as a result of acid prodictuction. aerobic production of acid from glycerol

Voges-Proskauer Test

Postive VP Test:

-color change to red, 2.3-butanediol fermentation (acetoin produced).

Negative VP Test:

-No color change, no 2,3 butanediol fermentation (acetoin is not produced).

Nitrate Reduction Test

determine whether an organism reduces nitrate to nitrite.

Endospore formers

Bacillus and Clostridium

Aeroduric anaerobes

resistance to oxygen even as they require anoxic conditions for growth

how to isolate endospore formers

add a gram of soil to TSY+glucose broth then heat in water bath to kill all the vegitative bacterial cells in the sample

how to create anoxic enviornment

Hydrogen gas reduces all of the oxygen inside of the jar to water resulting anaerobic atmosphere

Colostridium

gram positive Anaerobic GPROD, spore formers, exotoxin producers, catalase negative

Baccillus

large gram positive chain like rods, catalase positive

bergey's bacillus #1

contains pH indicators to indicate change in acidity. Purple to yellow to indicate glucose fermentation

Methyl Red Test

Measures the production of mixed acids by fermentation of glucose

Milk agar

Tests for the presence of the enzyme Caseinase, which hydrolyzes casein (a predominant protein in milk) into amino acid products. Casein gives milk its white color so a breakdown in casein causes the milk plate to lose its white color and become clear around the Caseinase positive colonies.

Starch Agar

Tests for the presence of Amylase, which hydrolyses starch to simple sugars. Iodine is added to starch plate and appears blue/black when interacting with starch. If amylase is present starch will be hydrolyzed and the blue/black color will not be observed around the amylase positive colonies.

Psuedomonads

gram negative, rod shaped protobacteria from soil, water, and plants. Oxidase positive

Oxidase test

identifies bacteria that have cytochrome oxidase

psuedomonads medium

minimal acetate or minimal asparagine broth

King's B agar

nutrient limited medium used to induce diffusable pigment production

gelatin deep

Hydrolysis of protein gelatin by gelatinase.

Once cooled to 4°C after incubation, liquefied medium indicates a positive result. Cooled solidified medium indicates a negative result.

Test may require up to 5 day incubation, or false negative results may occur.

Agar degrading bacteria

abundant in sea water and degrade agar for its carbon rich polysaccharides

ASN-III agar

used to enrich for agar degrading bacteria. contains only agar polymers

generation time (doubling time)

the time required for a complete fission cycle, from parent cell to two daughter cells

generation time equation

determine the amount of time taken to double the population

Acinetobacter

A gram-negative pathogen. naturally compitent and enriched using acetate. Prefer more acidic conditions.

Gram negative

oxidase negative

Enterobacteria

Gram negative rods that are frequently found in the intestinal tract

Catalase Test

Test that determines whether or not bacteria has "catalase" enzyme.

catalase enzyme

breaks down hydrogen peroxide into water and oxygen

shake tube: growth all across, uniform growth

aerotolerant

Colicins

proteins in Col plasmids that can kill neighboring bacteria

Nitrogen Reduction Test

used to identify organisms containing the enzyme nitrate reductase

Nitrogen reduction test positive