Bacteria overview

• Millions of bacteria live in small amounts of soil and water.

• Our gut microbiome contains more bacteria than cells (10*13 - 10*14)

  • 500-1,000 different species.

• Bacteria are extremely small, ranging from:

  • Archaea, which can be 0.4um in diameter, making them the smallest known organism which can reproduce independently.

    • This is only limited by the size of molecules necessary for life.

  • The largest is a sulphur bacterium, which is 750um in diameter and visible to the human eye.

    • Most of the cell is a vacuole.

Classifications

Shape

• There are three different shapes:

  • Coccus (spherical), such as Staphylococcus.

  • Bacillus (rod-shaped), such as E.coli.

  • Spiral (corkscrew-shaped), such as Spirillum.

• This can be further classified by their groupings:

  • Single, such as Helicobacter.

  • Pairs, such as Diplococcus pneumoniae.

  • Chains, such as Streptococcus.

  • Clusters, such as Staphylococcus.

Gram stain test

• This test allows scientists to distinguish between the composition of bacterial cell walls:

  • Positive bacteria remains purple throughout, due to its extra lipopolysaccharide layer.

  • Negative bacteria loses its purple and becomes pink, due to only having a thick peptidoglycan (murein) cell wall.

• This test happens in 5 steps:

  • Bacteria are inoculated, spread on a glass slide and heated. It is currently transparent.

  • Crystal violet (CV) is added to stain the bacteria. This binds to the peptidoglycan, staining all bacteria purple.

  • Lugol’s iodine is added to bind the CV to the peptidoglycan more strongly. This is known as a mordant (fixes a dye to a material). The bacteria remain purple.

  • Acetone-alcohol is added as a decolouriser, which removes unbound CV. Due to the lipopolysaccharide molecules, the purple dye washes off gram negative bacteria. Gram positive bacteria remains purple, and gram negative becomes transparent.

  • Safranin is added as a counterstain, in order to make the gram negative bacteria visible. The gram negative bacteria become red, and gram positive remain purple.

Gram stain positive

• This bacteria remains purple, and includes Staphylococcus and Streptococcus.

• The lack of a lipopolysaccharide layer in the cell walls allows the stain to bind effectively.

  • They are also more susceptible to antibiotics (such as penicillin) and the enzyme lysozyme than gram negative bacteria.

    • Penicillin works by preventing bonds between peptidoglycan from forming. This prevents new cell wall creation during mitosis, making the cell wall weak and allowing the cell to burst via osmosis.

    • Lysozyme is an antibacterial enzyme, in human tears and saliva, which works by hydrolysing bonds holding peptidoglycan molecules together.

Gram stain negative

• This bacteria loses the purple and becomes red, and includes Salmonella species and E.coli.

• This has a more chemically complex cell wall, as the outer cell membrane is supplemented with large lipopolysaccharide molecules.

  • These protect the cell from dyes, makes them penicillin resistant and unaffected by lysozymes.

• To control them requires antibiotics which interfere with the cell’s ability to make proteins.

  • These do not harm the eukaryotic (human) cells as they have different protein making cellular machinery.

Culturing bacteria

• Microorganisms undergo asexual reproduction and reproduce quickly in suitable environments.

  • In optimum conditions they can divide every 20 minutes.

  • This optimum can vary across bacteria.

Necessary conditions

• The following conditions are required:

  • Nutrients - Supplied using a nutrient media, either a liquid medium known as a nutrient broth or a medium solidified using agar. Mediums provide water and:

    • A carbon and energy source, usually glucose.

    • Nitrogen for amino acid synthesis, in organic and inorganic forms, such as nitrate ions.

  • Growth factors:

    • Vitamins, such as biotin.

    • Mineral salts, such as Na+ (sodium), Mg*2+ (magnesium), Cl- (chlorine), SO4*2- (sulphur), PO4*3- (phosphate).

  • Temperature - Enzymes regulate bacterial metabolism.

    • A range of 25-40 degrees is suitable for most bacteria.

    • Mammalian pathogens have an optimum of 37 degrees - human body temperature.

  • pH:

    • Most bacteria favour slightly alkaline conditions (7.4).

    • Fungi have an optimum of neutral to slightly acidic.

  • Oxygen need - There are 3 categories of oxygen need:

    • Obligate aerobes - Oxygen is required for metabolism, such as Mycobacterium tuberculosis.

    • Facultative aerobes - Microorganisms that do better with oxygen, but can survive in its absence, such as E.coli.

    • Obligate anaerobes - Microorganisms that respire without oxygen and cannot grow in its presence, such as Clostridium bacteria.

      • This produces toxins or poisons in a wound, destroying body tissue in a condition known as moist gangrene.

Aseptic technique

• This is also known as sterile technique, where all bacteria and apparatus are kept clear of microorganisms to prevent contamination of the bacteria culture but also the environment.

• To prevent culture and apparatus contamination:

  • All apparatus and media is sterilised before use to prevent initial contamination.

  • Culture vessels are flamed before opening and closing.

  • Sterile loops are used to prevent subsequent contamination.

• To prevent environmental contamination:

  • Work surface is sterilised before and after use using a disinfectant.

  • During inoculation (introduction of bacteria to encourage it to grow):

    • The culture bottle is held in one hand, and the cap is held in the other hand. It should not be placed on the work surface.

    • The mouth of the bottle should be flamed for 2-3 seconds.

    • Bunsen burners should be continuously heated as this causes bacteria to rise due to a convection current - therefore away from the apparatus.

    • The inoculation loop should be heated until red hot and then cooled.

    • The petri dish lid should be lifted just enough for the loop to enter.

      • There are two techniques for this, spreading (a pipette drop and then spread) and streaking (done using an inoculation loop. Three streaks are made, then a slight turn and three more. This is done five times, making a shape that resembles a hexagon.).

    • The lid should be secured with tap, but not all the way as this would close off oxygen, encouraging the growth of anaerobic and likely pathogenic microorganisms.

    • Incubate at 25 degrees, avoiding 37 degrees as this would encourage pathogenic microorganisms to grow.

    • Petri dishes should not be opened after incubation (when bacterial colonies grow).

  • In labs, autoclaves are used.

    • These are sealed containers where glass and metal equipment is heated to 121 degrees in steam under pressure for 15 minutes after the optimum pressure is reached.

    • Disposable materials after use, such as petri dishes, are sealed inside autoclavable plastic bags, autoclaved, then disposed of.

Measurements

• Estimating the growth of a bacterial population in a suspension (a fluid in which particles are dispersed but not totally dissolved) is important.

  • It is done by environmental health officers taking food samples.

  • Water authorities check water supplies daily.

  • Food manufacturers must check the food they sell.

• Food and drugs are produced using bacteria grown in industrial fermenters, and bacterial count is important.

• There are two methods:

  • Directly - Counting individual cells.

    • Viable counts only count living cells.

    • Total counts include living and dead cells.

  • Indirectly - Measuring the turbidity (cloudiness) of the culture.

Directly

• It is not possible to count all bacteria, so only a small sample is counted.

• Dilutions are also necessary in order to count all the bacteria due to high population density.

  • As this only counts bacterial colonies, only live cells are counted.

• Serial dilution is used, where a substance is diluted multiple times, usually of 1cm (moves up in 10^-1) or 0.1cm (moves up in 10^-2) suspension to 9cm or 9.9cm dilution.

  • 1cm of each diluted sample is spread over a sterile agar plate and incubated over 2 days at 25 degrees.

  • If a sample isn’t diluted enough, colonies merge (clumping) together and make counting inaccurate.

  • Only a dish containing 20-100 viable colonies that are distinct and separate are chosen.

    • This sample is then multiplied by the dilution factor.

• A haemocytometer can also be used, a more accurate method.

  • This is a specialised microscope, with a raised platform in the centre with perpendicular lines, allowing for individual counting.

  • However, this counts both living and dead cells.

Indirectly

• Turbidimetry is used, where a colorimeter is used to measure the turbidity of the culture, which would increase alongside the cell concentration.

  • This would measure the population by finding the exact absorbance of a suspension and comparing it to a graph of light suspension alongside bacterial cell count.

  • This provides a total cell count, as colorimeters do not distinguish between living and dead cells.

• This is used in the field, such as in river water samples.