Microbiology Lab Midterm

ubiquity

present nearly everywhere

Normal flora

microorganisms associated with animal body tissue

Nosocomial infections

infection originate in hospital environments

Colony

visible cluster of bacteria growing on solid medium, presumably cultured from a single cell

Colony formation

when one microorganism is deposited on an agar surface and the nutrients provided allow a cell to start dividing

Sterile

free of any living organism

Contamination

introduction of any unwanted organisms

TSA

Trypticase Soy Agar

TSB

Trypticase Soy Broth

Environmental sampling procedure

• obtain sample of microorganism using cotton swab

• zigzag inoculation

• dispose of cotton swab into biohazard bin

• Label date, initials, and sampling location on agar side of plate

• place agar side up in incubator

Why are plates stored upside down?

to prevent condensation on the agar

What should all be found on a correctly labeled dish

organism, date, initials, temperature

Where should the label be placed on a dish

around the outside edge on the agar side

For a tube, where should the label not be placed

near the lips of the tube

Colony morphology

• Shape

• edge

• elevation

Primary purpose of agar plate

isolation

Primary purpose of agar slant

storage

Primary purpose of agar deep

test oxygen requirement and motility

Primary purpose of agar broth

grow large amounts of cells

What does TSA have that TSB does not

agar

Aseptic technique

ability to transfer cells from one place to another without contaminating the original culture, the new medium, or the environment

Aseptic technique procedure

flame the instrument between before and after each use and flame the top of the tube

where should media tubes be placed when not in use

in a rack

Which two bacteria were used for lab 2 aseptic technique

E. coli and Bacillus subtilis

E. coli is a facultative

anaerobe

B. subtilis is an obligate

aerobe

What growth patterns can be identified on slants

• filiform

• arborescent

• beaded

• effuse

• rhizoid

• echinulate

Bacterial growth in broth

• pellicle

• sediment

• turbid

• floculent

Bacterial growth in deep

Who developed pure culture

Robert Koch

Mixed culture

a culture of many cells

Pure culture

one type of cell

Isolation streak procedure

Flame between each streak, inoculate plate, streak four times

Carrying microscope

use two hands and report any problems

How are images viewed in a microscope

reversed and inverted

Overal magnification

ocular lens x objective lens

What is the magnification for the ocular lens

10x

What are the magnifications of the objective lens

• 4x

• 10x

• 40x

• 100x

Resolution

the ability to distinguish between two points in an image

Contrast

the ability to distinguish between the sample and the background

Parfocal

the ability for a microscope to stay relatively in focus from one objective to the next

Storing microscope

• 4x objective lens in place

• oil should be cleaned from lens

• lower stage

• wrap cord

• slides removed

• place microscope on desk

Four eukaryotic microbes examined in Lab 4

• Protozoa

• fungi/yeast

• algae

• helminths

Protozoa

Paramecium

Helminth

scolex

Fungi

Aspergillus

Algae

volvox

all cells have a ___ charge

negative

A basic stain stains the ___ and an acidic stain stains the ___

cell; background

Chromophore

a colored ion

Basic stain means ___ chromophore

positive

simple stain means ___ chromophore

one

Bacterial smear preparations

1. small drop of water on slide

2. aseptically add and mix bacteria

3. allow smear to air dry

4. heat fix

Simple stain preparation

1. begin with heat fixed emulsion

2. cover with methylene blue

3. rinse with water

4. blot using bibulous paper

Which dye was used in the simple stain

methylene blue

A negative stain is acidic and therefore stains the

background

Steps to negative stain

1. Add nigrosin to slide

2. aseptically add organisms

3. use second slide to pull then push the dye

4. air dry

Which dye was used in the negative stain

nigrosin

what is the purpose of gram staining

differentiate between gram positive and gram negative organisms

Crystal violet

primary stain

Iodine

mordant

Ethanol

decolorizer

Safranin

counterstain

Gram staining steps

1. start with heat fixed sample

2. cover with crystal violet for one minute

3. rinse with water

4. cover with iodine for one minute

5. rinse with water

6. decolorize with alcohol for ~20 seconds

7. rinse with water

8. counterstain with safranin for one minute

9. rinse with water

10. blot with bibulous paper

Gram negative stains

pink

Gram positive stains

purple

Why is oil immersion needed for 1000x objective

to help focus the light by reducing refraction

What color does E. coli stain in gram staining

red

What color does micrococcus luteus stain

purple

E. coli is gram

negative

M. luteus is gram

positive

Defined media

exact chemical composition

Complex media

easy to use

selective media

selectively inhibit/foster growth

differential media

indicator dye to distinguish species

Mannitol Salt Agar (MSA)

selective for gram + and fermented mannitol turns plate yellow

Which organisms grew on the MSA

SA and SE

Which organism did not grow on the MSA

EC

Which organism turned the MSA plate yellow

SA

Which indicator dye is used in MSA

phenol red

Phenylethyl Alcohol Agar (PEA)

selective for gram + and used for isolation

Which indicator dye is used in PEA

none

Which species grew the best of PEA

SA and SE

Eosin Methylene Blue agar (EMB)

selective for gram -, ferments lactose, and recognize coliforms

What indicator dye is used in EMB

Eosin Y and Methylene Blue

Color of EC in EMB

metallic green

Color of PV in EMB

Pink

Color of EA in EMB

Pink

Color of SE in EMB

none

Is E. coli a coliform?

yes

MacConkey agar (MAC)

selective for gram -, fermentation of lactose cause change from colorless to red

What inhibits the growth of gram + in MAC

Bile salts

What is the indicator dye used in MAC

neutral red

Which two organisms grew on MAC

EC and EA

What color were EC and EA in MAC

pink

What instrument of sterilization have we used in most labs

Bunsen burner

____ ____ is a method used to transfer bacterial cultures from one place to another without contaminating yourself, the source, culture, or the destination culture

aseptic technique

Which medium did you inoculate with an inoculating needle

TSA deep

What is the purpose of the streak plate method

to get isolated colonies

What does it mean to heat fix a slide

to run the slide through the bunsen burner several times to stick the bacteria to the slide and kill it