Microbiology

The physical differences between bacteria relate to their size,cell wall structure and hence their staining characteristic and shape. These genetic differences produce different metabolic features and different surface molecules,causing different antigenic properties.

Different shapes:

Bacillus= rod

Spirillum = spiral

Coccus= spherical

Arrangement:

Strepto = chain/line

Diplo = two stuck together

Staphylo = random planes of division, sheets and clumps

Plasma membrane = barrier between environment and cytoplasm, controls entry andexit of substances in and out of cell,approx 7nm.

Cell wall = peptidoglycan/murein-preventslysisin a hypotonic solution.

Circular DNA= contains genes necessary for normal functioning of bacterium, protein replication.

Capsule = Outerlayer of mucopolysaccharide slime which can glue bacteria together, stick them to surfaces and protect the bacteria from attack by other cells.

Ribosomes = synthesis of proteins, 70S.

Pili = for attaching to surfaces and for transferring plasmids by conjugation.

Flagellum = For motility, (smaller and simpler than eukaryotic flagellum).

Plasmid = Circular DNA which contains extra bacterial genes including genes for antibiotic resistance.  Can be exchanged between bacteria during conjugation allowing the spread of antibiotic resistance.

Have no membrane bound nucleus, havelarge circularpieceof DNA in a region of cytoplasmcallednucleoidand many small circular fragments of DNA called plasmids.

Have no membrane bound organelles.

Smaller ribosomes than eukaryotes.

Cell wall made up of peptidoglycan (murein) - NOT cellulose

Generally very small 0.5-5 um (eukaryotes usually8-40 micrometres across)

They reproduce by binary fission.

Plant cell structure either: just cellulose, cellulose and lignin, cellulose and suberin / wax.

Examples: palisade (leaf), xylem, endodermis.

Fungi cell structure: chitin.

Examples: yeast.

Gram positive bacteria cell structure: Peptidoglycan - holds onto purple dye, isn't washed out by ethanol.

Examples: Streptococcus, Bacillus straphlococcus (MRSA) Penicillin is effective against them, inhibits enzyme for bond formation betweeb peptidoglycan fibres.

Gram negative bacteria cell structure: Complex cell wall withthin layer peptidoglycan and thick outer layer of lipopolysaccharide - crystal violet is removed when rinsed with ethanol as lipopolysaccharide layer is removed, then stains red / pink when counterstain safranin added.

Examples: E.coli, penicillin not effective, vancomycin instead.

Gram positive bacteria such as MRSA (staphylococcus), when gram stained appears purple. Has a outer cell wall of peptidoglycan and has no lipopolysaccharides.

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Gram negative bacteria such as E.coli turn red when gram stained. Has an inner, thin layer of peptidoglycan and has a think outer layer of lipopolysaccharide.

• Take a loop-full of bacterial culture from agar plate - don't lift lid fully off
• Mix into small drop of sterile water onto a clean glass slide
• Air-dry and heat-fix the smear by passing through the flame (2-3 times)
• Flood the heat-fixed film with crystal violet (primary stain) for about 30 seconds, then drain and wash with plenty of running water
• Add iodine stain for one minute before draiing and washing with running water
• Flood with ethanol (alcohol / acetone) for 30-45 seconds only
• Add safranin (counterstain) for 30 seconds, drain and wash under running water
• Gently blot in a tablet of bibulous paper, do not rub

Bacteria that take up crytal violet stain are stained purple, these are gram positive bacteria.

Peptidoglycan bound to by crystal violet, isn't washed out by ethanol.

Bacteria that take up the counterstain (safranin) are stained red/pink, these are gram negative bacteria.

Think outer lipopolysaccharide layer is originally stained with crystal violet, but is removed when washed with ethanol which dissolves lipopolysaccharide later. Counterstain is then used (safranin) which stains the thin peptidoglycan layer red/pink.

Physical

• Suitable temperature - depend on enzymes, usually alkaline
• Suitble pH - depend on enzymes, usually alkaline
• Oxygen requirements - respiration type

Nutritional

• Carbon source - usually organic e.g. glucose = energy source (depend on enzymes)
• Nitrogen source - inorganic = nitrate, ammonia - organic = amino acids. For building DNA and proteins (depend on enzymes)
• growth factors e.g. vitamin and minerals (inorganic ions), things bacteria can't manufacture

Obligate aerobes only respire aerobically, require oxygen

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Obligate anaerobes only respite anaerobically, grow in absence of oxygen

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Facultative anaerobes are abole to survive in absence of oxygen but grow and divide quicker in presence of oxygen.

Cover cuts so bacteria doesn't get into blood stream, as could cause disease / infection.

Don't eat or drinik in lab because food could be contaminated.

Wash hands thoroughly after experiment because bacteria could be on hands and don't want to transfer to other parts of body such as mouth.

(Not an aseptic tecnique but very important safety features)

Incubate cultures at 28 C not 37 C, as 37 C more pathogens can grow.

Secure the lids of Petri dishes with 2 strips of tape, don't seal as anaerobic bacteria that could be harmful could grow, pathogens grow well in anaerobic conditions. 

When opening the media or culture bottle, flame the neck and keep hold of the top as convection current pulls air out of bottle that could contain bacteria.

Heat innoculating loops to red heat in a bunsen flame, allow to cool before use so bacteria want isn't killed.

Heat glassware and media in an autpclave at 121 C for 15 minutes before use beause any bateria present before needs to be removed, glass is sterilized.

When accessing a Petri dish, only life the lid slightly as bacteria could easily escape otherwise.

Work next to a lit bunsen as creates rising air to prevent things falling into culture.

Swab down the bench with Virkon before carrying out an experiment, this removes bacteria already present on surface.

Heat inoculating loops to red heatr in a Bunsen flame after use, removes bacteria after stopping spread of bacteria.

Heat cultures and contaminated materical in an autoclave to 121 C for 15 minutes before disposal, this kills any remaining spores / bacteria.

Swab down the bench with Virkon after carrying out experiment to remove any bacteria that may have landed on surface during experiment.