Week 4 - Serial Dilutions & Unknown 1

Serial Dilutions =

A method of diluting an unknown concentration of bacteria in a series of known volumes for the purpose of calculating the unknown concentration.

IDF:

• Individual dilution factor

• Volume Transferred / (Volume Transferred + Volume Present)

What does this image show?

sample serial dilution - IDF

TDF =

• total dilution factor

• TDF will inc as you continue diluting

• IDF1 x IDF2 x IDF3 . . .

What does this image show?

sample serial dilution — TDF

CFU =

• colony forming unit

• unit commonly used to estimate the concentration of microorganisms in a test sample

• It is an isolated group of bacteria that is theorized to have originated from a single bacterium.

• The number of visible colonies (CFU) present on an agar plate can be multiplied by the dilution factor to provide a CFU/ml result.

To find the concentration of the stock solution:

CFU / (TDF x Volume)

• The units will be CFU/ml

• Range of viable colonies 30-300

• If a plate has more than this, it is called too numerous to count (TNTC). If a plate has less than this, it is called too few to count (TFTC)

What is on this slide?

calculation example

What method is this?

pour plate

What method is this?

spread plate

Selective media:

inhibits growth of some organisms while encouraging the growth of others

Differential media:

contains indicators to expose differences between organisms​

Undefined media:

contains one or more components with unknown composition​

Defined media:

all components are known​

Phenol Red Fermentation Test =

• Used to differentiate organisms based on their ability to ferment various carbohydrates

• Phenol red is a pH indicator which turns yellow below a pH of 6.8 and fuchsia above a pH of 7.4

If the organism is able to utilize the carbohydrate:

an acid by-product is created, turning the media yellow

When the organism is able to use the carbohydrate:

a gas by-product may be produced — If it is, an air bubble will be trapped inside the Durham tube — If the organism is unable to utilize the carbohydrate, gas will not be produced, and no air bubble will be formed

What does this show?

phenol red results

Bile Esculin Agar =

Bile esculin agar is a selective and differential medium which is used to identify organisms based on their ability to hydrolyze (break down) esculin.

BEA: Gram-positive bacteria other than some streptococci and enterococci are =

inhibited by the bile salts in this medium

If an organism can hydrolyze esculin:

the media will turn dark brown or black

What does this show?

bile esculine agar results

Catalase Test =

• Catalase is an enzyme produced by some species of bacteria

• This enzyme protects bacteria from hydrogen peroxide (H2O2) that can damage and kill them

• Catalase will convert hydrogen peroxide into liquid water (H2O) and oxygen gas (O2)

• Testing for the presence or absence of catalase can help identify bacteria

• A simple test to determine if bacteria produce catalase is to add hydrogen peroxide to bacteria

What does this show?

catalase test results

How to perform the catalase test:

• Use a sterilized loop to smear a small amount of bacteria onto a glass slide.

• IMPORTANT: do not use colonies from blood agar to run this test! Blood contains catalase and will give false positives.

• Place a drop of hydrogen peroxide (H2O2) onto the smear.

• Look for bubbles

Aerobic Bacteria =

• microorganisms that grow in the prescence of oxygen

• can only survive with oxygen

• live in soil, water, and on different surfaces

Aerobic Bacteria =

• microorganisms that grow in the absence of oxygen

• live in oxygen depleted areas such as the digestive system

Obligate Aerobes =

• microbes that require oxygen for growth

• They cannot survive without oxygen and are typically found growing at the top of a liquid media

Facultative Anaerobes =

can grow in the presence or absence of oxygen — does not use oxygen in any metabolic processes

Microaerophiles =

• survive in environments with low concentrations of oxygen.

• They need some oxygen to survive, but cannot tolerate large quantities of it

Aerotolerant Anaerobes =

do not require oxygen, but they are capable of growing in the presence of oxygen

Obligate Anaerobes =

grow strictly in the absence of oxygen — Oxygen is toxic to their survival

Thioglycolate is a medium designed to test:

aerotolerance in bacteria

4 main components of Thioglycolate Medium:

1. Yeast Extract

2. Sodium Thioglycolate

3. Methylene Blue

4. Agar

Yeast extract =

nutrient source for the bacteria

Sodium Thioglycolate =

reduces O₂ to water in the media

  O2 + H⁺ → H₂O (Oxygen Reduction Reaction)

Methylene Blue =

color indicator in the presence of O₂

Agar =

slows oxygen diffusion through the media

What does this show?

what growth of different aerobes/anaerobes look like