LAB EXAM BISC 303

Completed Exercises: 1, 2, 3, 4, 5, 6, 7, 8, 9

magnification of the oculars on the microscope

10X

Steps to establish Kohler

Fully wrack condenser up, diaphragm half open and adjusted for glare.

focus on specimen with 10X objective

close field diaphragm fully

slightly lower condenser until optimum focus

centre hexagon

open field diaphgram until lighted edge of hexagon just clears field of view.

how to establish phase contrast

establish kohler

switch to phase contrast objective

focus on specimen

push angular stop into position

increase light as needed

fine focus

inoculum

number of cells to be inoculated to form a bacterial culture

inoculated

cells were transfered to some medium to form a culture

3 main functions of a streak plate culture

1.Single colonies can be isolated and purified from an uncharacterized primary source (eg. soil, water, sputum etc.).

2. Colonies may be described on a morphological basis; morphological traits generally are expressed stably when environmental conditions stay the same.

3. Isolated colonies provide a source of pure inoculum for sub-culturing and starting further experiments.

size of each small line on stage micrometer

10um apart

chemoheterotrophs

use organic molecules as a source of carbon and energy

anaerobic or aerobic → oxygen is used as the terminal electron acceptor

the organism is aerobic

anaerobic or aerobic → If the organism is using an inorganic electron acceptor in an electron transport chain, that is not O2,

anaerobic respiration.

microaerophilic organisms

require free oxygen but at a very limited concentration

facultative anaerobes

can adapt to aerobic or anearobic conditions

role of sodium thioglycolate in media

promotes anaerobic growth. This compound reacts with oxygen and maintains enzymes in the cell in a reduced condition. Methylene blue is also added to indicate the presence of oxygen in the medium

drawing magnification

actual size of the drawing you made / actual size of the organism (measured via eyepiece)

purpose of Azide blood agar base

used to isolate and differentiate enterococci, which are resistant to sodium azide. supplemented with blood is also used to determine hemolytic properties of enterococci

role of agar deep tubes

test tubes filled with solid media that has been allowed to solidify vertically. Agar deeps are used to culture microorganisms that require less oxygen, such as anaerobic bacteria.

how a brewer anaerobic jar works (ones used in this lab)

several means. We use modern catalysts, that generate carbon dioxide as well as the hydrogen required to react with oxygen and create a strongly reducing environment.

Alternatively, air can be replaced with an inert gas.

what kind of bacteria is growing in thioglycolate broth tube 2

anaerobic bacteria

what kind of bacteria is growing in thioglycolate broth tube 1

aerobic bacteria

what kind of bacteria is growing in thioglycolate broth tube 3

a facultative anaerobe

Germicides

chemical agents that kill microorganisms

microstatic agents

inhibit the growth of microorganisms but do not kill them. Upon removal of the microstatic agent the microorganisms will resume their growth.

Disinfectants

may be either germicides or microstatic agents but are not considered safe for medical use and are applied only to inanimate objects. Household cleaning agents often contain disinfectants, such as ammonia and bleach (hypochlorite) to control the growth of microorganisms

Antiseptics

are similar to disinfectants but may be applied safely to biological tissues. These substances

are used for topical (surface) applications and are not necessarily safe for consumption. Examples

include alcohol, iodine and silver nitrate

The zone of inhibition technique and how it works

A filter disk is soaked in the disinfectant of choice and placed on the surface of a plate evenly spread with the bacteria of interest. If the disinfectant is effective, it will prevent the growth of the organism in a ring around the filter disk, referred to as the zone of inhibition.

In general, the larger the zone of inhibition, the greater the antimicrobial activity of the substance

The basic dye used in a Gram stain:

Crystal violet

the mordant used in Gram stain

Gram;s iodine

A mordant

a substance—typically a metallic salt—used to set and bind dyes to fabrics

decolourizing agent used in Gram stain

ethyl alcohol (95% Alcohol used in lab)

or an alcohol-acetone mixture

role of the decolourizing agent in a Gram stain

…decolourizes

used to wash unbound stain out of the sample

what happens to Gram Positive bacteria after decolourizing in Gram stain, why?

Gram-positive bacteria bind the dye strongly after treatment with the mordant (iodine) and retain the initial colour throughout the decolourizing procedure.

what happens to Gram Negative bacteria after decolourizing in Gram stain, why?

Gram-negative cells bind less strong to the original dye, so are decolourized and can be restrained with a counterstain of contrasting colour

result of the KOH test on Gram Negative cells and why

Gram negative cells are more permeable and can be lysed by 3% KOH. When the Gram negative cells are lysed, the DNA forms long sticky strands resulting in a viscous mixture

result of the KOH test on Gram Positive cells and why

Gram positive cells are less permeable and are not lysed by 3% KOH. because cells are not lysed, no sticky strands are formed.

How to prep a slide for a gram stain, if using a broth culture.

1. heat slide by passing through flame

2. label and draw a dime sized circle with a Grease pencil AKA the well

3. using aseptic technique, obtain a loopful of organisms, if not turbid try a drop from a pasteur pipette.

4. air dry smear

5. ONCE DRY, fix the smear by passing through the flame 2-3 times, should have a warm not hot slide.

How to prep a slide for a gram stain, if using a solid agar culture.

1. heat slide by passing through flame

2. label and draw a dime sized circle with a Grease pencil AKA the well

3. place a loopful of tap water on the slide

4. emulsify a small amount of culture into the drop, if the colonies are large, use cells from the outer edge of the colony.

5. air dry smear

6. ONCE DRY, fix the smear by passing through the flame 2-3 times, should have a warm not hot slide.

How to perform a Gram Stain

1. Fill well with Gram’s Crystal violet for 60 seconds, then tip off stain and rinse gently with water. remove excess water with wicking or shaking DO NOT WIPE!!

2. Flood Well with Gram’s Iodine for 60 seconds, then rinse with water, shake and wick.

3. Decolour with 95% alcohol for Approximately 10 seconds or until colour stops leaching from the slide. IMMEDIATELY rinse with water, gently blot away excess

4. counter stain with safranin for 10-20 seconds, rinse and blot off excess water

5. air dry

Endospores

tough, dormant, cell structures produced by certain bacteria like [Bacillus and Clostridium]. Resistant to Desiccation, UV light, hear and chemicals. can remain dormant for extended periods of time

what is A

Inner forespore membrane of an endospore

what is B and its role

Spore coat of an endospore (made of several protein layers). responsible for chemical resistance

what is C

Cortex (soft peptidoglycan)

what is D

Nucleoid

what is E

outer fore spore membrane

what is the position of endospore a

Central

what is the position of endospore b

subterminal

what is the position of endospore c

terminal

what is the position of endospore d

terminal with swollen sporangium

what cells produce endospores

vegetative cells

Sporangium

vegetative cell containing an endospore

what is used to stain endospores

Malachite green

what colour do endospores appear after staining technique using malachite green

Green

what colour do vegetative cells and the sporangium (not including endospore) appear after staining technique using malachite green

light red

how to perform an Endospore Stain

1. prepare a thin smear of culture on a clean slide, allow to dry, then fix with heat

In the fume hood

2. place a piece of bibulous paper (AKA filter paper) on the slide, just covering the film of the bacteria

3. SATURATE the paper with malachite green

4. heat slide over boiling water on a hot plate for five minutes, adding more stain if it starts to dry.

5. dispose of the paper in the waste container, cool the slide and gently rinse with water.

at the lab sink

6. counterstain with safranin for 30 seconds

7. rinse with water and blot dry

Selective media 

contains an inhibitor which slows/prevents growth of some organisms without affecting the growth of the organisms you want to s

what does Maconkey’s agar do

inhibits Gram positives, is used to pick up Enterobacteriaceae (E. coli and cousins). 

also, lactose fermenters will have pink colonies.

manitol salt media

inhibits many bacteria which are not salt tolerant, allowing certain Gram positives to grow unimpeded, especially Staphylococcus.

alsoe S. aureus (an important pathogen) ferments mannitol and produces a yellow colour.

differential media

contains ingredient(s) that make one type of organism appear different from others. 

what happens to lactose fermenters on macConkey’s agar

form pink colonies

what happens to S. aureus on mannitol salt agar

ferments mannitol and produces a yellow colour

 Blood agar,

made with fresh blood containing whole cells, is used to differentiate between different types of Streptococcus, based on their ability to form a zone of clearing by lysing the red blood cells (called hemolysis).

Enriched media .

contain special ingredients that allow extremely fastidious organisms to grow

chocolate agar

made with autoclaved blood the media preferred by Neiserria gonorrhea and Neiserria meningiditis, two important pathogens that look like Branhamella in a Gram stain.