Lab Practical

What are some simple stains?

methylene blue, carbol fuschin, crystal violet, safranin

What does the ocular lens do?

The eyepiece you look through (usually 10x magnification)

What does the objective lens do?

Provides different magnifications (4x, 10x, 40x, 100x)

How do you calculate total magnification?

Ocular magnification × objective magnification

study the parts of the microscope

What color are Gram-positive bacteria after staining?

purple

What color are Gram-negative bacteria after staining?

pink/red

What stain is applied first in Gram staining?

crystal violet

What is the counterstain in Gram staining?

safranin

types of differential staining

gram staining and acid fast staining

gram staining

positive or negative

acid fast staining

binds only to dangerous bacteria that have waxy material in cell walls

Special stains

endospore, flagella, capsule

Capsule staining

stains protective outer capsule of bacteria

negative stain—stains the background

Endospore

highly resistant to staining because they are a survival mechanism for bacteria, so they require special stain

Flagella

thicken appearance of flagella bc they are hard to see

Vibrio, spirillum, spirochete

Types of morphology

coccus, bacillus, spiral (vibrio, spirillum, spirochete)

What is the difference between nutrient broth and nutrient agar

Nutrient broth is in the test tube while nutrient agar is on the plate

Preserving bacteria cultures

deep freezing & freeze drying

deep freezing

-50→ -95 C

freeze drying

-54 → -72 C

How many biosafety levels are there?

1,2,3,4

BSL 1

basic teaching labs

BSL 2

lab coat, gloves, eye protection

BSL 3

air filters and ventilation due to infectious airborne pathogens

BSL 4

space suits, government facilities

Thermal death point

lowest temperature in which all cells in a liquid culture are killed in 10 minutes

Thermal death time

mimimum amount of time it takes for all bacteria to be killed at a certain temp

What does an autoclave do?

it kills bacteria and inactivates spores using moist heat (steam) and denaturing their proteins

What are the requirements of an autoclave

121 C

15 PSI (pressure)

autoclave tape must show sterile

Types of pasteurization

High temperature short time (HTST), Ultra high temperature (UHT)

HTST requirement

72 C 15 seconds

UHT requirements

140 C 4 sec

Dry heat

for instruments that can’t really handle heat

170 C

2-3 hours

Filter sterilization

for passage of things through a screen

HEPA filters

Types of radiation

ionizing and nonionizing

Nonionizing radiation

ultraviolet (UV)

Nonionizing radiation—UV characteristics

creates thymine dimers

cant penetrate glass and plastic

UV lamps

hospital safety

Ionizing radiation

X-rays, gamma rays, electron beams

Ionizing radiation characteristics

can penetrate glass and plastic

causes OH radical damage to DNA

food safety

Zone of inhibition

disk diffusion method

area around a chemical agent that was disinfected

shows it works

What are the four phases of bacterial growth?

Lag, Log, Stationary, Death

What happens in the lag phase?

bacteria adjust to environment

What happens in the log phase?

rapid bacterial growth and division

What happens in the stationary phase phase?

the growth rate equals the death rate

What happens in the death phase?

more bacteria die than reproduce

What environmental factors affect bacterial growth?

temperature, environment, pH, oxygen

Bioguanides

effective against gram +, many gram -, & enveloped viruses

Bacteriocin

proteins that produce one bacteria that inhibit another

flagella staining

mordant and carbol fuschin

endospore staining

malachite green, heat, water, safranin

capsule staining

india ink/nigroskin—background, simple stain for actual bact

acid fast stain

carbol fuschin, heat, decolorize, methylene blue