Microm 302: Practical Exam

True or False:

Most bacteria species are not harmful and they do preform useful and indispensable functions for life

True

What's the purpose of agar?

It's a good solidifying agent since it has a high melting point (100 C) and low solidifying point (~45 C)

True or False:

Bacteria is everywhere and makes it hard to have sterilized equipments and media

True

What's the correct way to label a plate?

name

date

sample

procedure if relevant

Why do we invert plates?

-To prevent condensation on bacterial colonies

-water will provide mobility to bacteria and prevent them from forming colonies

Why do the samples vary in numbers of colonies from the Ubiquity of Bacteria lab?

There are different colonies growing depending on the sample source

Will every microbes present in a sample grow and form colony?

No, some cannot grow because the environment might not be right (oxygen level, nutrients, ect.)

What factors influence growth of bacteria?

The amount of nutrients on media, space,

How to carry microscopes correctly?

-Carry it upright

-Don't rest it on body while walking

-Don't bump it into locker and lab bench

Ocular lenses (eye piece) magnify objects ___

10X

Objective lenses magnify objections ___ (4)

4X, 10X, 40X, and 100X

To calculate the total magnification...

ocular x objective

stage adjustment knobs are used to....

adjust the position of the slide on the stage

iris diaphragm

adjusts the amount of light

coarse focus knob

focus image in 4X or 10X objective lenses

fine focus knob

minor focusing adjustments for 40X and 100X only

lamp brightness control dial

use to adjust microscope light

width of pointer in 10X objective

25 um

width of pointer in 40X objective

6 um

width of pointer in 100X objective

2.5 um

1 um = ___ m = __ inches

10^-6 m

1/256000 inches

Protozoa size

20 - 100 um

Yeast size

4 - 10 um

Bacteria

1 um

Virus

0.1 um (too small to be seen with light microscope)

How to use Darkfield

-open iris diaphragm completely or else dark field will block all light, giving you a dark field (haha funny joke)

Dark field does NOT work in ____ objective

100X

Wet mounts can be used for (5)

-determining size

-morphology

-grouping

-motility

-general type and other characteristics

How to do a wet mount

add drop of liquid and put on cover slide

Brownian motion

motion due to atoms bombardment

looks like random vibrations

Motility

directional movement of individual cells

"swimming"

What magnification should the lenses be for safe storage?

4X

Aseptic technique (2 parts)

-anything that will come into contact with culture need to be sterilized

-need to be sterilized before the object is set down on the counter or for disposal

Where is the hottest part of the flame?

Near the tip of the flame

Why should you not touch the glass when taking a loopful of bacteria out from a tube?

The loopful will be released to the side of the glass

Why should you wait until the loop is fully cool until you touch broth or agar?

-To avoid killing bacteria and creating aerosols (easily inhaled droplets that contain bacteria)

Why is one end of the pipette is plugged with cotton?

To filter incoming air and to prevent material from floating to the top

Green pipet aid are used for ____ and ___ mL

5 and 10 mL

True or False

You can touch the sterilized pipet to the side of the glass of the tube.

False: anything that touches the pipet (except broth) will result in contamination

True or False

Pipet must be place into the waste container directly after touching bacteria. (You can't place it on the counter)

True

True or False

Cloudiness = turbidity = bacterial growth

True

Why do we use stains?

To make their shape and groupings more visible

(colorless bacteria are hard to see)

True or False

Staining kills bacteria

True

So we don't need to boil slides before washing or discarding

Staph. epidermis is a __ shaped bacterium

spherical /coccus

Citrobacter Freundii is a ___ shaped bacteria

rod

Spirillum voltuans is a __ shaped bacteria with distinctive ___

spiral shaped

volutin granules (metachromatic)

How to make a stain from solid sample?

-add loopful of water

-use sterilized loop to add bacteria

-spread to make a turbid circle

Why do you only need a very small amount of culture for staining?

-Harder to distinguish morphology

-cannot stain uniformly with thick smear

How to make stain from broth culture?

-transfer loopful of bacteria with sterilized loop onto slide

True or False:

You should spread the bacteria around on the slide if you use a broth culture.

False!!

Do NOT spread the bacteria or add water since broth culture are already diluted

What's the purpose of heat fix?

Make bacteria stick to the slide (or else it will easily slide off)

What dye should you use to see the volutin granules?

methylene blue

Basic dyes carry __ charge so they __ negative compounds on cell surface.

positive, attracted

Acidic dyes carry __ charge so they __ negative compounds on cell surface

negative, repel

Why should you dry the slide completely before doing oil immersion?

Water and oil don't mix, will distort image

Resolution

ability to distinguish two pints as separate

True or False

Humans have resolving power of 0.2mm

True

Anything that's 0.2mm or more apart will be seen as separate

Light microscope has resolving power of ___

0.2 um

True or False

Cover slides are only used for wet mount.

True

True or False

It is critical to keep oil from other lenses that are not 100X

True

Gram stain is an example of ___ stain

differential

Staph, epidermis is a Gram __ __ (shape and class-action)

Gram positive cocus (purple)

Citrobacter freundii is a Gram ___)

Gram negative rod (pink)

Gram stain procedure

crystal violet: 30 - 60s

iodine 30 - 60s: complexes crystal violet

decolorizer: 20 - 30 s

safarin 30 -60 s

True or False

Both Gram positive and Gram negative bacteria will look purple after it added crystal violet and after iodine

True

True of False

If done correctly, Gram negative should be colorless after decolorizer

True

How can both Gram negative and Gram positive be purple or both be colorless after decolorizer?

Both purple: not enough time for decolorizer

Both pink: too long with decolorizer

True or False

Gram stain are best done with young culture (12-24 hours) and it will retain stain for (less than 1 day minimum)

True

Colony

visible mass on media

Pure culture

a population descended from a single cell

True or False

streak plate gives pure cultures

True

Streak plate purpose

to dilute bacteria on media so they will only grow in isolated colonies

-can also distinguish color and shape

True or False

Closer streak lines dilute bacteria more evenly

True

Sulfonamides are unique because they display ____

selective toxicity (kill or inhibit bacteria without killing host)

bacteriostatic not bactericidal

How does sulfonamides only effect bacteria?

Bacteria: PABA --> folate --> coenzyme F

Human: folic acid from diet --> folate -- coenzyme F

* prevents PABA to folate

Sulfa drugs ____ inhibit PABA because their structures are ___

competitively inhibit

analogues/very similar

How to reverse PABA inhibition effects caused by sulfa

-add extra PABA to out compete Sulfa

-add amino acids to skip folate synthesis

Glucose tube only (SULFA)

control: to make sure bacteria can grow (positive control)

glucose + sulfa

no growth

glucose + sulfa + PABA

growth (shows bacteriostatic)

glucose + sulfa + TSY

growth because nutrient provided in media for bacteria to skip folate synthesis

Inhibiting growth of bacteria can treat UTI because...

your immune system can catch up after growth is inhibited

obligate aerobes

-need O2 to survive

-generate H2O2

-usually produce catalase to break down H2O2

obligate anaerobes

-kill in O2, don't need it

-no catalase

example is botulism bacteria

facultative anaerobes

will use O2 for metabolism if available

-produce catalase

example: E Coli

microaerophilic

need O2 but only tolerate small amount

aerotolerant anaerobe

grow in O2 but not used in metabolism

Why do you boil agar in the aerobic/anaerobic growth experiment for 10 mins?

TO drive off O2 diffused in agar

Obligate Aerobes (M. Luteus) grow on the ___ of tube

top, near dissolved O2 (if there's any)

Obligate anaerobes growth ____ of tube

bottom

facultative anaerobe grow __ of tube

everywhere

True or False:

Vegetative cells can be killed by boiling

True

True or False

Bacillus and Clostridium can produce endospore and resist heat, acidity, drying, irradiation

True

Endospores grow in response to ____

depleting nutrients or adverse conditions (sporulation)

True or False

E Coli does not produce endospores

True

Why do we incubate tubes with bacillus and E coli (heat sensitivity) after heat treatment?

To see if any cells survived and grow

Validate plate count must contain ___ and __ colonies

30 -300

Bacillus is killed at

100 C

E coli is killed at

75 C

True or False

UV light can't sterilize glass and dish because it can't penetrate glass or most substances?

True