BISC 303 Lab Exam

Kohler Illumination

1. fully rack up the condenser

2. focus on specimen with 10x

3. close the diaphragm (hexagon)

4. lower the condenser to until image is in optimal focus (strong hexagon)

5. center the hexagon

6. open diaphragm just enough so the light covers the whole field of view

phase contrast

use for unstained living organisms

1. set up kholer

2. push annular stop in

3.increase the light if needed

4. use fine focus

oil immersion

only use for 100x

1. focus on sample at lower x using kholer

2. rotate so that the oil immersion lens is almost over the specimen

3. add oil to the slide and rotate the lens on so it touches the oil

4. focus with fine adjustment away (

microscope maintenance

-use lens paper only

functions of a streak plate

1. to isolate single colonies to be characterized

2. to describe colonies on a morphological basis

3. source of pure inoculum for sub-culturing

micrometer

1000uM long

absolute smallest tick at 10x=10uM

absolute smallest tick at 40x=2.5uM

absolute smallest tick at 100x=1uM

aseptic technique

-keep things 10-20cm close to the flame (except EtOH)

-don't keep things open longer than necessary

-clean spills with hyamine or bleach soaked paper towels

-vortex a broth culture before using it

Different Broth Recipes

nutrient broth: beef extract + peptone

glucose mineral salts broth: salts + sugar

tryptic soy broth: tryptone, soytone, dextrose, salts

semi-enriched broth: glucose mineral salts + 1/10 nutrient broth

Anaerobic Culture Methods

-boil for 10 minutes and seal with wax/mineral oil

-using reducing agents such as sodium thioglycolate with methylene blue indicator

-brewers anaerobic jar:

a) H + O2 reaction catalysed by heated platinum or unheated palladium. excess water absorbed by CaCl2

b) distilled water + package containing catalyst

c) replace air with inert gas (vacuum needed)

disinfectants on microorganisms

germicides: kill microorganism specifically or not

microstatic: inhibit growth but wont kill them, upon removal they will grow again

disinfectants: may be germicides or microstatic agents, not safe for medical use and only are used on inanimate objects

antiseptics: like disinfectants but are applied on biological tissues

soap&water: reduces number of microorganisms on skin surface

alcohol: denatures proteins, extracts membrane lipids, dehydrating agent (antiseptic)

iodine (antiseptic): kills all types of bacteria, including spores

heavy metals (antiseptic): eg silver nitrate

detergents: cationic or anionic, eg quaternary ammonium salts

gram stain

1. heat slide by passing it through flame

2. draw a circle with a GREASE PENCIL

3. add a loopful of culture to the circle and spread out

4. dry under the flame

5. fix slide by passing it through flame 2-3 times

(wear gloves)

6. add grams CRYSTAL VIOLET and wait 60s, rinse with water (wick with paper towel but don't wipe)

7. add GRAMS IODINE and wait 60s, rinse with water and wick

8. rinse with 95% EtOH and rinse with water for ~10s or until it runs clear

9. add SAFRANIN for 10-20s

10. air dry

KOH test

add a drop of KOH and add a lot of bacteria from a plate. Gram negatives with have a mucoid thread

typical arrangements

single, pairs, chains, clusters, tetrads, palisades (dominoes)

endospore stain

1. prepare smear as with gram stain

2. soak bibulous paper with malachite green and lay over smear over boiling water for 5 minutes

3. counterstain with safranin for 30s

endospore arrangements

central

subterminal: not in the middle but not at the end

terminal

terminal w/ swollen sporangia

Flagellar Arrangements

monotrichous (polar, just 1 at one pole)

lophotrichous (many at 1 pole)

amphitrichous (1 at both poles)

peritrichous (all over)

determining bacterial motility

1. observe movement on 0.4% (semi-solid agar) inoculated in 1 position

2. observe swarming on 1.5% agar

3. wet mount: let sit for 10 minutes, view using phase contrast

4. use a mordant (ryu flagella stain) to make flagella visible

selective media

inhibits the growth of certain microbes while allowing other to grow (eg using crystal violet to inhibit G+ to grow)

differential media

reagents permit observer to differentiate between different types of bacteria (colour, morphology, ability to lyse RBC)

enriched media

adds additional nutrients such as blood or serum to a complex base; used for cultivating fastidious organisms

MacConkey Agar

- Bile Salt, Crystal Violet -> decrease G+ (selective)

- Identifies G- enteric bacilli

- Lactose fermenters: pink colonies from lowered pH (indicator)

Azide Blood Agar Base

sodium azide inhibits all species except streptococcus

also contains protein

Blood agar

same as blood agar base but also contains sheep blood

EMB Agar

Eosin Methylene Blue Agar (EMB)

contains protein, buffer, and a sugar

has eosin Y and methylene blue to inhibit G+

fermenters of the sugar lower the pH causing dye to ppt around them

-E. coli becomes metallic green

-non-fermenters are usually colourless

Mannitol Salt Agar

contains protein

D-mannitol (carbohydrate fermented by staphylococcus aures

phenol red (yellow in acid)

7.5% NaCl (inhibits most bacteria except for staphylococci)

oxidase test

detects cytochrome c oxidase

uses paper soaked in tetramethyl-p-phenylenediamine dichloride (TMPD), present if it turns purple when touching the bacteria

bacteria may need to be treated with DMSO to allow the TMPD to get in

catalase test

detects presence of catalase (decomposes H2O2 into water and O2 gas)

put the bacteria into a drop and look for the bubbles

gelatinase

detects hydrolysis of gelatin (collagen)

- innoculate a gelatin broth tube and incubate, if once put on ice, the gelatin solidifies, the test is negative

amylase

streak a starch containing plate with the bacteria and incubate overnight. the next day, pour iodine over the plate. if there is a halo around the growth, then the bacteria has starch amylase

IMViC series

Indole, Methyl Red, Voges-Proskauer, Citrate

used for the identification of Enterobacteriaceae organisms

indole production

detects the presence of tryptophanase by adding tryptophan. If Indole is created, when Kovac's reagent is added a red product will be produced

if glucose/phytone is added, acid production of the carbohydrate may inhibit tryptophanase by protein sparing action of carbohydrates

Voges-Proskauer Test

detects acetoin in intermediate of the fermentation pathway that leads to the production of a 2,3 butanediol

red colour develops upon addition of Barritt's reagents A and B that detect acetoin

methyl red test

Measures the production of mixed acids by fermentation of glucose

-run on MR-VP media and culture must be 48 hours

-positive is red, yellow is negative

Citrate Test

This test is used to determine if an organism uses citric acid as its SOLE carbon source

using Simmon citrate agar

utilization of citrate results in a royal blue colour

growth throughout is also positive

fermentation of carbohydrates

may produce acidic end products or gas

use pH indicator that is yellow when acidic (phenol red)

inverted tube to detect gas formation

dissimilatory reduction of nitrates by nitrate reductase

NO3 to NO2-

use media containing nitrate and test for nitrite

If nitrite is present, maroon colour develops when adding sulphanilic acid solution and dimethyl-alpha-naphthylamine

if no colour, it may have been reduced past nitrite into NO, N2O, or N2. Add zinc (turns nitrate into nitrite) and if it turns, red, the test is positive.

triple iron sugar agar

contains protein, salt, lactose (lots), sucrose, dextrose, sulfur, H2S indicator, pH indicator

glucose fermentors produce variety of acids turn media yellow. More acid is formed within the butt (fermentation) than the slant

red slant/yellow butt: glucose fermenter but unable to ferment lactose and sucrose

form of whole colony

punctiform, circular, filamentous, irregular, rhizoid (branched), spindle (elongated)

elevation

flat, raised, convex, pulvinate, umbonate (irregular)

margin (edge)

entire, undulate (bumpy), lobate, erose/serrate filamentous, curled

surface

smooth, rough, wrinkled, shiny

consistency

mucoid/slimy, dry, powdery, ropey, waxy

m-Endo agar

coliforms will be metallic green or dark red, pink or clear are not coliforms

R2A agar

dry, low nutrients, grows heterotrophic bacteria

dilution factor

old volume/new volume (eg 1 in 10)

OD600 = 0.1 is

2e8 cfu/mL

using the spectrophotometer

light beam must pass through 1cm, arrow on the cuvette points in the direction of the laser

readings below 0.05 can be unreliable

hemocytometer

setting up the mixture: 1 drop of methylene blue, vortex, transfer drop to the edge of the coverslip

the second smallest square (with 16 smaller squares inside) has a volume of 4nL, count non-blue cells

used 400x for yeast, 1000x for bacteria

MALDI-TOF

use a young culture and spread over the spot of the target plate, apply matrix to ionize proteins with laser, use database to identify species. G+ may need formic acid added

balanced growth

regular doubling of all cell components

conjugation

F cells if they have F plasmid (fertility factor), contains tra gene for F pilus

if it is integrated into the host genome, it is Hfr

Antibiogram

profile of antimicrobial sensitivity

Minimum inhibitory concentration (MIC)

the minimum concentration of a substance necessary to prevent microbial growth

agglutination

Clumping of microorganisms or blood cells, typically due to an antigen-antibody interaction.

should be a balanced proportion and Ag should be large

Ouchterlony

Double diffusion assay using a semisolid medium where adjacent samples of antigen and antibody solution interact to reveal identity, nonidentity or partial identity.

if the same antigen is placed in adjacent wells w/ antisera in the centre, bands will form a continuous line

if there are two different antigens, two independent lines will form a double spur

if two antigens that have some of the same components , only one spur will form towards the weaker antigen