Microbiology Practical 2 Theory

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Questions and Answers

What is the main usage of Baird-Parker agar?

Inhibition of yeast growth

Detection of staphylococci (correct)

Detection of E. coli

General microbial enumeration

Which ingredient in Baird-Parker agar helps to enhance the growth of staphylococci?

Peptone

Sodium chloride

Polypeptone

Glycine (correct)

What color change indicates a reduction of tellurite on Baird-Parker agar?

Grey to black (correct)

Yellow to red

Colorless to pink

Green to blue

What is the final pH of MacConkey agar?

7.1 Signup and view all the answers

What should be avoided when streaking bacteria onto selective and differential media?

Cross-contamination Signup and view all the answers

Which organism is specifically tested on Baird-Parker agar for coagulase positivity?

Staphylococcus aureus Signup and view all the answers

What temperature is recommended for incubating both MacConkey and Baird-Parker agar plates?

37°C Signup and view all the answers

Which ingredient in MacConkey agar serves as a selective agent against gram-positive bacteria?

Crystal violet Signup and view all the answers

What is the primary function of selective media in microbiological experiments?

To favor the growth of certain organisms while suppressing others Signup and view all the answers

Which component of MacConkey agar acts as a selective agent?

Bile salts Signup and view all the answers

What characteristic allows bacteria that ferment lactose on MacConkey agar to be visually identified?

Change in color of the pH indicator Signup and view all the answers

Which method is primarily used for the enumeration of bacteria in laboratory processes?

Spread Plate technique Signup and view all the answers

What is a defining feature of differential media in microbiology?

They provide observable differences in colony morphology Signup and view all the answers

In the context of selective media, what should be considered when choosing a medium for an experiment?

The growth characteristics of the target organism and the source material Signup and view all the answers

What additional material is included in the laboratory for the Personal Hygiene experiment?

Dye Reduction test Signup and view all the answers

What characteristic effects on the immediate environment can differential media reveal?

Variations in colony size and morphology Signup and view all the answers

What is the purpose of bile salts in MacConkey Agar?

To selectively inhibit non-enteric bacteria. Signup and view all the answers

In the serial dilution method, what is the dilution factor used when mixing 1 ml of sample with 9 ml of diluent?

10-fold dilution Signup and view all the answers

What is the primary purpose of using Nutrient Agar plates in the enumeration of bacteria from milk?

To detect bacteria present in diluted samples. Signup and view all the answers

During the enumeration of bacteria, how is the original concentration calculated after counting colonies?

By multiplying the number of colonies by the dilution factor. Signup and view all the answers

What is the purpose of using the spread plate technique in bacterial enumeration?

To obtain a uniform distribution of bacteria on the agar surface. Signup and view all the answers

What indicates a positive result for lactose fermenters on MacConkey Agar?

A color change to pink. Signup and view all the answers

Why is hand washing emphasized in the study of personal hygiene and transfer of microorganisms?

It can reduce the transfer of microorganisms significantly. Signup and view all the answers

What is typically the result of touching an agar plate with unwashed gloved fingers?

An increased number of colonies reflecting contamination. Signup and view all the answers

In the context of bacterial counts from a milk sample, what does CFU/ml stand for?

Colony Forming Units per milliliter. Signup and view all the answers

In which situation would you expect to see pink colonies on a MacConkey Agar plate?

When lactose fermenters are present. Signup and view all the answers

Study Notes

Lecture 9: Theory Behind Practical 2

The practical sessions include:
- Streaking bacteria onto selective media (Baird Parker and McConkey Agar) using the Streak Plate technique.
- Enumerating bacteria using the serial Dilution Method followed by the Spread Plate technique.
- A personal hygiene experiment.

Extra Online Material

Most Probable Number (MPN) test for water sampling
Membrane filtration
The Dye Reduction test

Selective and Differential Media

Many media are developed to select or differentiate between organisms.
Selective media: favour the growth of certain organisms while suppressing the growth of others.
- The choice depends on the organism and the sample's nature.
Differential media: show differences between bacterial species through colony morphology like size, colour, or influence on the surrounding environment.
- Baird-Parker agar and MacConkey agar are selective and differential examples.

McConkey Agar

A common selective and differential media for isolating and differentiating coliforms and other intestinal bacteria in water, dairy, and biological specimens.
Key ingredients: Lactose, peptone, bile salts.
Lactose fermenters produce a localized drop in pH, which, with neutral red as an indicator, creates dark red colonies.
This agar allows the isolation and differentiation of lactose-fermenting, bile salt-tolerant bacteria.
Composition:
- Peptone: 17 g/L
- Polypeptone: 3 g/L
- Lactose: 10 g/L
- Bile salts: 1.5 g/L
- Sodium Chloride: 5 g/L
- Agar: 13.5 g/L
- Neutral red: 0.03 g/L
- Crystal violet: 0.001 g/L
- Distilled water: 1 L
- Final pH = 7.1

Baird-Parker Agar

Used for detecting and enumerating staphylococci, particularly coagulase-positive organisms like S. aureus.
Widely used in food safety applications.
Ingredients include: lithium chloride and tellurite to inhibit other organisms, glycine, and pyruvate to enhance staphylococcal growth.
Tellurite reduction produces a grey-to-black colour and coagulase-positive organisms show a clear halo around the colonies.
Composition:
- Casein Peptone: 10 g/L
- Meat Extract: 5 g/L
- Yeast Extract: 1 g/L
- Lithium Chloride: 5 g/L
- Glycine: 12 g/L
- Sodium Pyruvate: 10 g/L
- Agar: 15 g/L
- Final pH (at 25°C): 6.8 ± 0.2

Streaking Bacteria

Use the streak plate method to inoculate MacConkey and Baird Parker agar, with provided organisms, while avoiding cross-contamination.
Incubate at 37°C.
Take photos and note observations after 48 hours for practical report.
Each organism is streaked on half of each plate. Example organisms: Staphylococcus aureus and Escherichia coli.

Enumeration of Bacteria (Serial Dilution Method)

Add sample (milk or otherwise) to an appropriate diluent (diluting solution - often water).
Repeat n dilution steps adding fresh diluent following each step.
Spread different dilutions across the agar plates.
Incubate the inoculated plates.
Count the colonies on the plates.
Calculate the concentration of bacteria in the original sample by taking the count, the plating factor, and the dilution factor into account.

Spread Plate Technique

Isolate colonies of interest visually.
Count bacteria colonies.
Use a sterile glass spreader to spread bacteria evenly on the surface of the agar plates to make colonies visible.

Enumerating Bacteria in Milk

Create a 10-fold dilution series of milk samples.
Use Nutrient Agar plates to identify bacteria in 10⁻², 10⁻³, and 10⁻⁴ dilutions.
Calculate the number of bacteria per millilitre in the original sample.

Personal Hygiene Experiment

The experiment involves the transfer of microorganisms through tissue paper.
It demonstrates the concept of hand washing preventing microorganism spread.
Different plates are handled by gloved and ungloved hands to observe differences.

Dye Reduction Test

Methylene blue is used as a redox indicator for Streptococcus lactis.
If Streptococcus lactis is present, the blue color will reduce indicating presence and quality of milk.
Poor quality milk will decolorise within two hours; high-quality milk retains color.

Membrane Filtration

Uses cellulose acetate or cellulose nitrate filters with pore sizes of 0.45 - 0.2 µm to separate bacteria.
Trapped bacteria can be viewed after transfer to an appropriate medium.
Quick, useful for field testing and higher reproducibility of results.

Most Probable Number (MPN) Method

Coliform bacteria in faeces/water can be determined via MPN.
MPN method involves diluting samples to different concentrations.
Inoculating these dilutions into different tubes.
Incubating at appropriate temperature.
Counting positive samples in each dilution series
Using a table to assess the total amount of coliform bacteria in 100 mL of sample.

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Description

This quiz covers the theoretical principles behind Practical 2 in microbiology, including methods for streaking bacteria and enumerating them through various techniques. It also delves into selective and differential media, particularly Baird Parker and McConkey agar, for studying bacterial growth. Explore the concepts of personal hygiene tests and additional water sampling methods as well.