6.1 - Microbial techniques

Why are aseptic techniques important when culturing organisms?

To produce uncontaminated culture, so results are reliable & repeatable 

What are the basic aseptic techniques?

• Wipe surfaces with antibacterial cleaner/disinfectant

• Set up Bunsen burner nearby & work underneath → convection currents prevent microbes from entering culture

• Flame inoculating loop & neck of bottles before use

• Minimise time that vessels containing bacteria are open

• Sterilise all equipment (e.g. using an autoclave)

• Wear protective clothing

How are microorganisms cultured?

1. Transfer bacteria to agar plate using sterile inoculating loop or pipette

2. Tape lid on at 2 ends, then invert the dish & incubate → ensure the dish is not airtight & don’t incubate above 25 °C to avoid the growth of pathogens

What is the difference between a spread plate & a streak plate?

• Spread plate: distributes microorganisms evenly with a sterile spreader

• Streak plate: aims to obtain single colonies by rotating the plate to build layers of the culture on at least 3 separate streaks

What is a broth culture medium?

Liquid nutrient medium where microorganisms have been introduced & allowed to multiply → results in a cloudy suspension of microbial cells

What is an agar medium?

Solid growth medium made from agar (jelly-like substance from algae) that contains nutrients:

• used to grow & culture microorganisms (e.g. bacteria) in the laboratory

What is a selective medium?

A kind of media that has a very specific set of nutrients to allow particular pathogens or bacteria to grow

What are the advantages of using broth & agar media?

Broth media:

• can provide anoxic & oxic conditions depending on the depth, which helps identify microbes/determine their optimum conditions

• can grow a very large volume of bacteria 

Agar media:

• can obtain a single, discrete pure colony for study 

What are the 4 phases of a bacterial growth curve?

1. Lag phase

2. Log phase

3. Stationary phase

4. Death phase 

What happens during the lag phase?

The population size increases slowly as the bacteria are acclimatising to the conditions (e.g. temperature, food & pH)

What happens during the log phase (exponential growth) ?

Growth is very rapid as the conditions are optimum for the bacteria → there are no limiting factors

What happens during the stationary phase?

• Number of bacteria remains constant as nutrients begin to run out & toxins start building up

• Reproduction rate is equal to the death rate  

What happens during the death phase?

Number of bacteria decreases as they die due to the build-up of toxic waste products & lack of nutrients (e.g. build-up of CO₂ & low pH)

What are the 3 methods to estimate the growth of a bacterial culture?

• Haemocytometry

• Turbidometry

• Dilution plating

How is haemocytometry conducted?

1. Dilute the broth sample with an equal volume of trypan blue to stain the dead cells blue

2. Use a calibrated haemocytometer with volume 0.1mm³ & count the cells in each set of squares & calculate the mean

3. Number of bacterial cells = number counted x 10⁴ per cm³ 

What are the advantages & disadvantages of using haemocytometry?

Advantages:

• quick → don't have to wait for microorganisms to grow or incubate 

• able to use it alongside turbidometry to provide reference values 

Disadvantages:

• provides total cell count

• large margin for human error

• difficult to get viable cell count unless a dye is used 

• cannot look at growth rates easily 

• requires a very good quality microscope 

How is turbidometry conducted?

1. Use a colorimeter & measure the absorbance or % transmission of samples with a known microorganism count

2. Plot a calibration curve → absorbance or % transmission (y-axis) against number of microorganisms (x-axis)

3. Record the absorbance or % transmission of the unknown sample & interpolate the graph

What are the advantages & disadvantages of using turbidometry?

Advantages:

• quick → don’t have to wait for microorganisms to grow or incubate 

Disadvantages:

• requires calibration curve from known samples

• provides total cell count

• easily contaminated → dipping into the culture frequently  

How is dilution plating conducted?

1. Grow a colony from a single microorganism

2. Perform a serial dilution with distilled water to see single colonies

3. Prepare a lawn plate & count the colonies

4. Number of cells = number of colonies x dilution factor

What are the advantages & disadvantages of dilution plating?

Advantages:

• doesn't require machinery/automatic devices to do a cell count 

• provides viable (living) cell count

Disadvantages:

• slow → incubation period needed

• have to work aseptically, meaning time is required to thoroughly sterilise all of the equipment 

• requires expensive equipment

What is the equation to calculate the growth rate constant (k) & what do the terms mean?

• N_t = the number of organisms at time t

• N₀ = the number of organisms at time 0

• k = the exponential growth rate constant

• t = the time for which the colony has been growing