Lab 2

Question 1

Colony

Answer 1

Visible growth created by multiple rounds of bacterial reproduction (cloning)
*each colony represents a single cell

Question 2

Aseptic?

Aseptic technique?

Disinfect?

Sterile?

Contaminate?

Answer 2

Aseptic -free of living pathogens

Aseptic technique - set of procedures to prevent contamination

Disinfect - to reduce microbial numbers to safe levels

Sterile - complete removal of all microbes, including spores

Contaminate - to introduce unwanted microbes

Question 3

What aseptic techniques do we follow in our lab?

Answer 3

1. Disinefect work area
2. Wash hands after cleaning the bench
3. Properly sterilize inoculating loops and needle

*Only bring culture out when need it
*don’t work over top of items
*Dispose of things properly

Question 4

Pure culture?

Mixed culture?

Contaminated?

Answer 4

Pure culture - Culture containing only 1 bacteria

Mixed culture - Contains more than 1 type of organism

Contaminated - unwanted organisms as well as desired organism

Question 5

Streak plate technique

Answer 5

common pure culturing technique

thins out small inoculation of bacterial cells over the surface of the entire plate

*separate individual cells from one another that will form colonies, which will consist of clones

Question 6

Staining

Answer 6

Process of adding contrast to microbes

Question 7

Positive stain

Simple stain

Differential stain

Answer 7

Using positively charged chromogens (basic) that adhere to bacterial cells (negative charge)

Simple stain - use a single chromogen to color cells the same color

Differential stain - uses 2 or more chromogens based on their biochemical composition

Question 8

Smear prep

Answer 8

Label slide

Plate culture - 1 drop of saline on the slide, transfer a small amount of plate growth to the drop and swirl it

Broth culture - mix the culture and transfer 1-2 loopfuls to the slide

Allow to air dry

Gently heat - hold glass slide in front of incinerator 5-6 times

let cool

Question 9

Gram stain

Gram-positive

Gram-negative

Answer 9

A differential stain that uses various dyes

Gram-positive - a bacterium with a thick peptidoglycan layer and no outer layer

Gram-negative - a bacterium with a thin peptidoglycan layer and outer membrane

Question 10

Gram stain process

Answer 10

1. Prepare a smear of bacteria
2. Apply chrystal violet, 30 sec and wash off
3. Apply grams iodine. Mordant. sets stain. 1 min
4. Decolorize with grams alcohol 2-5 sec
5. Counterstain with Safranin. 30sec to 1 min. wash
6. Blot to remove excess water and air dry

Question 11

beta-hemolysis (β-hemolysis)

alpha-hemolysis (α-hemolysis)

gamma-hemolysis (γ-hemolysis)

Answer 11

beta-hemolysis (β-hemolysis) - beta -complete lysis, the clear area around colony growth

alpha-hemolysis (α-hemolysis) - alpha - incomplete lysis of red blood cells, green area around colony growth

gamma-hemolysis (γ-hemolysis) - gamma - no red blood cell lysis

Question 12

The 3 basic shapes of bacteria

Coccus -

Bacillus -
Vibrio-
Coccobacillus -

Spirillum -

Answer 12

Coccus - round or spherical

• *Bacillus** - rods
• *Vibrio**- curved rods
• *Coccobacillus** - short rods

Spirillum - spirals, corkscrew

Question 13

Common mistakes for gram stains

Answer 13

1. smear prep too thick
2. Over/under decolorizations
3. Culture age, if over 16-18hours

Question 14

Mordant

Answer 14

Forms an insoluble chemical complex

a substance, typically an inorganic oxide, that combines with a dye or stain and thereby fixes it in a material.

Question 15

What is the purpose of flamming the culture tubes prior to entry with loops?

Answer 15

to produce convection currents to prevent microbes from the air from entering the culture

Question 16

What will disinfection of the bench kill?

not kill?

Answer 16

Will kill most vegetative cells and viruses but will not destroy endospores

Question 17

What is degerming?

Sanitization?

Disinfecting?

Sterilizing?

Answer 17

Degerming - hand washing

Sanitization - to reduce or eliminate pathogenic agents (such as bacteria) on the surfaces of (something)

Disinfecting - reduce microbes

Sterilizing - complete removal of microbes

Question 18

How does an autoclave work?

Answer 18

Medical tools and equipment are placed inside an autoclave. The lid is sealed, air is removed from the autoclave, and then steam is pumped into the vessel. Heat and pressure are maintained long enough to kill microorganisms and bacteria in order to sterilize medical tools.

Question 19

What is one major difference between serratia marcescens and Escherichia coli?

Answer 19

E coli - either motile or non motile, no red pigment

Serratia marcescens - motile and red pigment

Question 20

What color is gram positive?

What color is gram negative?

Answer 20

Gram positive is purple

Gram negative is pink

Question 21

Describe the gram stain reagents and their purpose?

Answer 21

Crystal violet - stains both positive and negative peptidoglycan layers

Grams iodine - mordant that forms an insoluable chemical complex with crystal violet

Grams alcohol - removed chrytsal violet iodine complex from gram negative making them colorless. Shirnks pores of gram positive trapping the dye. Decolorization

Safranin - stains decolorized gram negative pink to red.

Question 22

why is the gram stain a differential stain?

Answer 22

used to differentiate between gram positive organisms and gram negative organisms.

Question 23

Why is the decolorization step the most important?

Answer 23

it can remove color and give false positive or negative

Question 24

why is the gram stain the most important in first step of IDing a unknown bacteria?

Answer 24

allows for the determination of the Gram reaction, morphology, and arrangement of the organism.

Question 25

What clinical info does the gram stain provide?

Answer 25

it helps your doctor learn if you have a bacterial infection, and it determines what type of bacteria are causing it. This can help your doctor determine an effective treatment plan.

Question 26

Why does culture age affect the results of the gram stain?

Answer 26

Old cultures tend to lose the peptidoglycan cell walls, which predisposes gram-positive cells to be gram-negative or gram variable.

Question 27

What are the causes for false-positive and/or false-negative

Answer 27

False-positive - clumping of dye

False-negative - too much washing, too much alcohol