microbioLAB #3: Basic Lab and Culture Techniques

What is sterilization?

Sterilization is the process of

rendering a medium or material free of all forms of life.

What are the Five different techniques?

1. autoclave

2. dry-heat sterilization

3. ultraviolet (uv) radiation

4. ethylene oxide gas

5. filtration

autoclave

is a use of pressure chamber to sterilize

equipment supplies oat 121 degree and 15 lb of pressure

for 15 min or longer of pressure, conditions under which

microorganisms will not survive, even endospores

Dry-heat sterilization:

- glassware such as pipettes and petri plates are placed in an

electric oven at 160 -170 degrees for 2 hours.

3. Ultraviolet (UV) radiation:

what can it not sterilize?

- Use of radiation at 260 nm to sterilize air and exposed surfaces.

- UV can’t be used to sterilize glass, water, and other substances

4. Ethylene oxide gas:

- Microbicidal and sporicidal.

- It is used for heat-sensitive material such as plastics

5. filtration:

• Bacteriological filter when media is made from components that will not withstand heating at high temperature.

• Bacteriological filter removes bacteria and larger microorganisms from the solution using a membrane with 0.22 mm pores.

Microbiological Media

what is media?

what are the physical forms of media?

What is the purpos for each?

Media : nutrient preparations that are used for culturing

microorganisms.

Three physical forms of media:

1- Liquid or broth

• to propagate large numbers of microorganisms.

2- Semisolid contains Agar

• to determine bacterial motility and promote anaerobic growth.

3- Solid contains Agar

• a) for surface growth of microorganisms in order to observe colony appearance

• b) for pure culture isolation

• c) for storage of cultures

• d) observe specific biochemical reactions

Making solid media

In a liquified state, media is poured into

1. Test tube for agar slants and agar deep tube.

2. Petri dish- for agar plate.

3. Liquid media-broth tube, this shows turbid solution of bacterial cell growt

Liquid agar + test tube = slant/deep
Liquid agar + Petri dish = plate
Liquid media WITHOUT agar + tube = broth

And turbid = cloudy, which means bacteria have grown in the broth.

look at image for making solid media

Microbiological Media

• what are the two diff media used to culture microorganisms?

Two different media used to culture microorganisms

1- Chemically defined or synthetic - known amounts of pure

chemicals

2- Complex or non synthetic media - complex materials rich in vitamins and nutrients usually beef extract, yeast extract and

peptones

how is sterile media made?

When lab personnel make media they measure out

a quantity of dry powdered nutrient media, add

water and check the pH.

 They dispense the media into bottles, cap it and

autoclave. This is a process similar to home canning

techniques in food preservation.

 The autoclave exposes the media to high

temperature (121°C) and pressure (15 psi) for 20

minutes.

 Once the media is autoclaved (or pressure cooked)

it is considered sterile (all life forms killed)

Bacteria and other microbes have particular requirements for growth. in order to successfully grow bacteria in lab,we must provide an environment suitable for growth. HOW/what do u need?

whats media?

whats TSA?

• Growth media (singular = medium) are used for microbial growth.

• Media = mixtures of nutrients that the microbes need to live.

  • Also provides a surface and the necessary moisture and pH to support microbial growth.

• Tryptic Soy Agar (TSA) is the media that we often use

  • Complex nutrient media which supports the growth of a wide variety of microbes

Culture Transfer Instruments and Techniques

pipette

inoculating loop

Pipette: instrument used to transfer aliquots of culture.

 Pipettes ranges 20-200 ul and 100-1000 ul

•  Down to the 1st level then 2nd level to eject.

 Inoculating loop: transfers microorganisms from 1 media to

another.

how to use micropipettes

spread plate technique:

- Label with a Sharpie the BOTTOM of the Agar plate.

- Pipette a 100 ml volume of a bacterial mixture

onto the center of an agar plate.

- Spread evenly over the surface with a sterilized spreader until the liquid

is absorbed.

- Invert the plate and incubate at 37 degrees for 24-48 hours

spread plate technique

 Bacterial mixture is transferred to the edge of an agar plate

with an inoculating loop and spread in a specific pattern.

 This creates a diluted gradient (most colonies in the 1st

section to the least in the last).

 pure culture Can be developed from colonies well isolated

contaminatin of a streak plate is caused by what?

leaving the plate open too long or not shielding properly with the lid

whats the purpose of streaking? principle?

• purpose—to obtain pure, isolated colonies

• principle—by spreading a large amt of bactria over the large surface of a plate, the amt of bacteria is diluted until individual cells are spread on the surface of the plate. Ea individual cell grows into a single colongy. all of the cells in this colony are genetically identical

• Ea species of bacteria produces a distinctive colony appearance.

Streaking = spreading bacteria out to separate individual cells.

Individual cell → grows into a colony → colony contains genetically identical bacteria → gives you a pure culture.

the pour plate technique is used for what?

bacteria and fungi

in pour plate technique the original sample is diluted many times because?

diluted many times to yield isolated colonies

pour-plate method

Culturing: serial dilution

Serial dilution- lowering the concentration of substance in a

stepwise fashion or a series of dilution. The dilution is constant

between each step. Helps to quantify them amount cells in culture

Dilution equation for culturing: serial dilution

Dilution = (vol transferred/final vol) x initial dilution

1. To convert from uL to mL

2. To convert from mL to uL

1. divide by 1000

2. multiply by 1000

Dilution Calculation Example:

1st dilution is 50 μl of undiluted bacterial culture is added to 4.95 ml of saline solution

2nd dilution 50 μl of 1st dilution (10-2) is added to 4.95 ml of saline solution

FIRST DILUTION

undiluted bacteria = 50 uL bacteria

add 50 uL to 4.95 mL saline

1) first convert 50 uL to mL

50 ÷ 1000 = 0.05 mL

2) find total volume to find final volume

4.95 mL + 0.05 mL = 5.00 mL

3) Use the dilution formula

= (vol transferred)/(final vol) x initial dilution

= (0.05)/(4.95+0.05) x 1←undiluted culture

= 0.01 =10^-2 ←100-fold dilution

SECOND DILUTION

= (0.05)/(4.95+0.05) x 0.01←fr first dilution

= 0.0001 = 10^-4 ←10,000-fold dilution

final plate should have between how many colonies?

25-250

number of colonies = what?

= number of live bacteria that grow = CFU colony forming units

• CFU/ml = number of colonies in the plate x dilution factor