3.3 Microbiology

Why do we learn about Bacteria?

Immunology, Gut Health, Medicine, Food Production.

• Plasmids
• Nucleoid
• Ribosomes (70s)
• Cell Membrane
• Peptidoglycan Cell Wall.

Bacteria can be grouped by;

1. Shape

2. Staining (Gram Stain)

3. Phylogenetics (Evolutionary Relationships)

4. Type of disease they cause.

Single Sphere- Coccus (s), Cocci (P)

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Chain of spheres- Streptococci

Bunch of Spheres- Staphylococci

Rod - Bacillus

Spiral - Spirillum

Naming Bacteria

Genus+Species, Bacteria have Binomial Names.

Cross between a Coccus and a Bacillus= Coccobacillus

Gram Stain

Differential Stain that stains the cell wall.

Gram Positive- Deep Purple Colour, stains the thicker less permeable peptidoglycan.

Gram Negative- Pale Red Colour, stains the thinner more permeable membrane.

1. Take a sample of Bacteria, and make a smear.

2. Heat fix the bacteria to the slide.

3. Stain with crystal violet.

4. Stain with Iodine. - All bacteria take on the purple stain.

5. Decolourise with ethanol.

6. Counterstain with safranin.

   

Structure of the Cell Wall

• In Gram Positive Bacteria; Very thick peptidoglycan
• In Gram Negative; Lipopolysaccharide membranes, a thinner peptidoglycan layer.
• Alcohol removes the outer LPS membrane and because the peptidoglycan layer is thinner, it is more permeable and crystal violet washes out.
• Gram Stain is a quick test to give patients antibiotics very quickly.

29/11/2022

Starter

1. Bacillus
2. Coccus
3. Thinner peptidoglycan layer, more permeable membrane, LPS membrane
4. Thick Peptidoglycan layer, therefore less permeable cell wall, therefore crystal violet stain not removed during decolourisation step with ethanol.

Why would you want to know the number of bacteria living in a culture, and how would you work it out?

• Bacterial cell count- find out use by date, water contamination levels, adding cultures to milk & cheese.
• How to work it out- Respiration???

Counting bacteria: Viable Counts

Viable- Living bacteria.

Direct Counts- Counting the number of cells, can be either a viable count or a total count.

Indirect Counts- Using turbidity (cloudiness) to estimate the number of bacteria. Uses a calibration curve.

Viable Count- If we want to know the number of bacteria per cm^3.

Produce a series of dilutions, each sample is more diluted.

Plate a known volume of dilution onto plates,

Where a bacteria falls, a colony is produced.

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Number of colonies X Dilution Factor = no of bacteria cm-3

Volume Plated (cm^3)

Eg ; 152x10^5 = 3.04x10^7

0.5

1. 45x10^2 = 4.5x10^4

0.1

1. 128x10^6 = 1.28x10^9

0.1

1. 40x10^3 = 4.0x10^5

0.1

Aspetic Technique

1. Prepare serial dilutions
2. 1cm^3 of orginal culture to 9cm of sterile dilutent.
3. Plate out known volume on to nutrient agar plate
4. Count colonies.

06/12/2022

Viable-living cell

Viable cell count- Aseptic technique, count clumps,

(No of Colony x dilution factor)/ volume plated.

Total Count

Haemocytometer- Microscope slide with counting grids

No of cells per cm^3 = No of cells in counting area x dilution factor

volum e in counting area (cm^3)

Mm to cm = / by 1000