Biology: Microbiology

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Classification of Bacteria

There are said to be a million bacteria in 1cm3 of fresh water and 40 million in 1g of soil. The human body has 10^13 cells but living in the gut our "microbiome" contains 10^14 bacteria, of 500-1000 different species. It has been estimated that worldwide there are 5x10^30 bacteria and that their combined mass is greater than the combined mass of all animals and plants. These huge numbers alone make it seem sensible to study bacteria.

The physical differences between bacteria relate to their size, their cell wall structure and, hence, their staining characteristics and their shape. Their genetic differences produce different metabolic features and different surface molecules, so they have different antigenic properties.

Bacteria vary considerably in size. The smallest are Archaea, such as Nanoarchaeum equitans, at 0.4 um in diameter, is the smallest known organism that can reproduce independently. The largest bacterium described is the sulphur bacterium Thiomagarita namibiensis. It is 750um diameter and visible to the naked eye, although most of the cell is a vacuole. E. Coli is 1.8um diameter and 7 um long.

Haemophilus influenzae is one of the smallest bacteria known. It is important as it is found in the human respiratory tract, and was the first organism to have its entire genome sequenced.

Classification by Shape

A genus of bacteria has one of three main shapes, and the shape is sometimes indicated in its name:

1. Bacillus or rod-shaped, e.g. Escherichia; Bacillus

2. Coccus or spherical, e.g. Staphylococcus; Streptococcus

3. Spiral or corkscrew-shaped, e.g. Spirillum.

Further differentiation is often possible according to the way bacteria tend to group. They may be single, e.g. Helicobacter; in pairs, e.g. Diplococcus pneumoniae; in chains, e.g. Streptococcus; or in clusters, e.g. Staphylococcus.

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Classification by the Gram Stain Reaction

The Gram stain allows microbiologists to distinguish between Gram-positive and Gram-negative bacteria. The different staining properties are due to differences in the chemistry composition of their cell walls. Before staining, bacteria are colourless. After staining Gram-positive bacteria are stained violet and Gram-negative bacteria are red.

Gram stain: a method of staining the cell walls of bacteria as an aid to their identification.

There were three major advances in microbiology between 1881 and 1887: Fanny Hesse introduced agar jelly into the laboratory, Hans Gram invented his stain and Julian Petri invented his dish.

Gram stain binds to the peptidoglycan of bacterial cell walls. Archaea and Eukaryotes do not contain peptidoglycan and are not stained by Gram stain.

The cell wall of all bacteria is a 3-dimensional network of polysaccharides and polypeptides, known as peptidoglycan or murein. The cross-linking of these molecules provides strength and gives the cell is shape. The wall protects against swelling and bursting or lysis caused by the osmotic uptake of water. Gram-positive bacteria possess this basic cell wall structure and Gram-negative bacteria have an additional outer layer of lipopolysaccharide.

Gram staining has four stages:

1. Crystal violet - basic dye - binds to peptidoglycan so all bacteria stain purple

2. Lugol's iodine - Mordant - binds to crystal violet to the peptidoglycan more strongly

3. Acetone-alcohol - Decolouriser - removes unbound crystal violet and lipopolysaccharide

a. gram-negative bacteria lose their stain and become colourless

b. gram-positive bacteria remain purple

4. Safranin - Counter-stain -

a. gram-negative bacteria stain red

b. gram-positive bacteria remain purple

Gram-Positive Bacteria

After staining, Gram-positive bacteria are violet or purple under the microscope. They include Bacillus, Staphylococcus and Streptococcus.

The absence of an outer lipopolysaccharide layer in the cells walls of Gram-positive bacteria allows them to bind stain efficiently and makes them more susceptible to the antibiotic, penicillin, and the enzyme, lysozyme, than Gram-negative bacteria.

Bacteria constantly makes and break chemical links in their cell walls. The antibacterial enzyme lysozyme, which occurs in human tears and saliva, hydrolyses the bonds holding the peptidoglycan molecules together.

Penicillin prevents the bonds inter-linking peptidoglycan molecules from forming. This is especially significant when the bacteria make new cell walls when they divide. Penicillin therefore makes the cell walls structurally weak and prone to collapse. Water uptake by osmosis bursts the cell.

Gram-Negative Bacteria

Gram-negative bacteria have a more chemically complex cell wall than Gram-positive bacteria. Their outermost membrane is supplemented with large molecules of lipopolysaccharide which protect the cell and exclude dyes like crystal violet, so they appear red or pink in the microscope.

Gram-negative bacteria include Salmonella species and E. Coli. The outer lipopolysaccharide protects the peptidoglycan below and so they are not affected by lysozyme and are resistant to penicillin. To control them requires a different class of antibiotics, that interfere with the cell's ability to make proteins. Eukaryotic cells also make proteins, but the protein-making cellular machinery is different from that in bacteria, so these antibiotics do not harm them.

Animal cells do not have a cell wall, so human cells are not damaged by penicillin.

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Conditions Necessary for Culturing Bacteria

Micro-organisms can undergo binary fission and reproduce quickly, given a suitable environment. In optimum conditions they divide every twenty minutes. In the laboratory, bacteria can be grown on a wide variety of substrates providing they are supplied with nutrients, water and suitable physical conditions, such as temperature. Micro-organisms vary in their requirements but usually grow over a range of temperatures and pH values, with an optimum within the range.

Micro-organisms require the following conditions for growth:

1. Nutrients - in the laboratory, nutrients are supplied in nutrient media. The bacteria may be cultured in a liquid medium, called a nutrient broth, or on medium solidified with agar. The media provide water and they include:

a. A carbon and energy source, usually glucose.

b. Nitrogen for amino acid synthesis, in organic molecules and in inorganic form, such as nitrate ions.

2. Growth factors including vitamins, e.g. biotin and mineral salts e.g. Na+, Mg2+, Cl-, (SO4)2-, (PO4)3-.

3. Temperature - as bacterial metabolism is regulated by enzymes, the range of 25-45C is suitable for most bacteria. The optimum for mammalian pathogens is around 37C, the temperature of the human body.

4. pH - most bacteria are favoured by slightly alkaline conditions (pH 7.4), whereas fungi grow better in neutral to slightly acid conditions.

5. Oxygen - many micro-organisms require oxygen for metabolism and are obligate aerobes, e.g. Mycobacterium tuberculosis. Some grow best in the presence of oxygen, but can survive in its absence; these are facultative anaerobes, e.g. E. Coli. Others cannot grow in the presence of oxygen and are obligate anaerobes. Clostridium bacteria are obligate anaerobes that produce toxins or poisons in a wound. They destroy body tissue in the condition called "moist gangrene".

There are different ways to describe culture media.

1. A "defined" medium contains only known ingredients.

2. An "undefined" medium contains components that are not all known, because they include, for example, yeast extract or beef peptone.

3. A selective medium only allows certain bacteria to grow, e.g. MacConkey agar only allows Gram-negative bacteria to grow; media containing tetracycline only allow tetracycline-resistant bacteria to grow.

Many bacteria cannot be cultured because they may be: mutualistic, needing other species, intracellular parasites, have very particular growth requirements, have a very long generation time or be poisoned by media components.

Two potential problems which must be avoided when working with bacteria are the contamination of cultures from the environment and the contamination of the environment from the cultures.

Principles of Aseptic Technique

Bacteria and fungi are cultured on or in media that are designed to supply the cell with all its nutritional and physical requirements. Aseptic technique, also known as sterile technique, in which the apparatus and equipment are kept free of micro-organisms, prevents contamination of bacterial cultures by other microbes and contamination of the environment.

To prevent the contamination of pure cultures and apparatus by bacteria from the environment:

1. Sterilise all apparatus and media before use to prevent initial contamination.

2. Handle cultures carefully, flaming the necks of culture vessels before opening and closing and use equipment such as sterile loops to prevent subsequent contamination.

To prevent contamination to the environment by the bacteria being used in experiments:

1. Sterilise the work surface before and after an experiment using a disinfectant, for example, 3% Lysol or Vercon.

2. Use the correct handling techniques to prevent the contamination of personnel and the immediate environment by the organisms being cultured. When carrying out the process of inoculation:

a. Hold the culture bottle in one hand; remove the cap with the little finger of the other hand. Do not place the cap down on the work surface.

b. Flame the mouth of the bottle for 2 or 3 seconds.

c. Pass the inoculating loop through a flame until it is red hot, and allow it to cool in the air.

d. Lift the lid of the Petri dish just enough to allow entry of the inoculating loop.

e. Secure the Petri dish with two pieces of adhesive tape. Do not seal all the way round as this could create anaerobic conditions and potentially, encourage the growth of pathogenic micro-organisms.

f. Incubate at around 25C. Cultures should not be cultured at 37C as this is an ideal temperature for the growth of many pathogenic species.

g. Do not open Petri dishes after incubation.

3. In a laboratory the preferred method of sterilisation is to use an autoclave. This is a sealed container in which glass and metal equipment is heated at 121C in steam under pressure for 15 minutes after the required pressure has been reached.

4. After use, disposable materials, such as plastic Petri dishes, can be sealed inside autoclavable plastic bags, autoclaved and then placed in a dustbin.

Gamma radiation is used commercially to sterilise plastic equipment that would melt in an autoclave.

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Methods of Measuring Growth

Estimating the growth of a population of bacteria is extremely important. Environmental health officers regularly inspect food premises and take samples for analysis. Water authorities check water supplies daily. Food manufacturers must ensure the food they sell is fit to eat. Many items, such as foods and drugs, are produced using bacteria growth in 200dm3 industrial fermenters. Accurately measuring their population growth is an important part of the process.

The size of a population of micro-organisms in liquid culture may be measured:

1. Directly, by counting cells.

a. Viable counts describe living cells only.

b. Total counts describe living and dead cells.

2. Indirectly, by measuring the turbidity (cloudiness) of the culture. This method can be used as a fieldwork technique, using light absorption by a sample of river water to indicate the number of bacteria present.

Measuring Growth Directly

Plating and Counting Colonies

It is not possible a whole population of micro-organisms. Instead, the number of cells in a very small sample of culture is counted. Even then, the population density is likely to be so high that cell counts have to be made using dilutions of the culture. This type of count relies on each live cell forming a colony, so it provides a viable count.

Colony (of bacteria or fungus): a cluster of cells, or clone, which arises from a single bacterium or fungal spore by asexual reproduction.

The sample is diluted using serial dilution. When 1cm3 suspension is added to 9cm3 medium, it has been diluted 10 times and is a 10^-1 dilution. If this process is repeated, dilutions of 10^-2, 10^-3, 10^-4 and so on can be made. But if 0.1cm3 is progressively added to 9.9cm3, the first dilution is 10^-2 and subsequent dilutions are 10^-4, 10^-6, 10^-8, e.t.c.

1cm3 of each diluted sample is spread over a sterile agar plate and incubated at 25C for two days, allowing the bacteria to grow. A dish containing 20-100 colonies that are distinct and separate is chosen and the colonies counted, To find the total viable cell count, the number of colonies is multiplied by the appropriate dilution factor.

If a 1cm3 of a 10^-5 dilution produces a mean of 85 colonies per plate:

the initial concentration = 85 x 10^5 = 8.5 x 10^4 cm-3.

If 0.5cm3 of a 10^-7 dilution produces a mean of 129 colonies per plate:

the initial concentration = (129/0.5) x 10^7 = 258 x 10^7 = 2.58 x 10^9 cm-3.

If the dilution is too great, there will be too few colonies on each plate for the count to be statistically sound. If the dilution is insufficient, colonies merge, referred to as "clumping", and counting may be inaccurate, resulting in an underestimate of the numbers.

The separate colonies of bacteria are counted assuming that each colony has arisen from a single cell from the original culture which has divided asexually.

Using a Haemocytometer

This is a more accurate method than colony counting. It uses a specialised microscope slide, called a haemocytometer. It is not possible to distinguish between living and dead cells so the result is a total cell count.

Growth rates can be estimated by regularly measuring the diameter of a bacterial or fungal colony spreading from a central point over the surface of a solid growth medium.

Measuring Growth Indirectly

Turbidity

A colorimeter can be used to measure the cloudiness or turbidity of the culture as cell numbers increase. Measurements of the bacterial population are derived by finding the absorbance of the suspension and then reading from a standard graph of light absorbance plotted against the number of bacterial cells. The result is a total cell count, because the colorimeter cannot distinguish living from dead cells.