Flashcards in Microbiology Deck (28):
How many bacteria are said to be in 1cm^3 of freshwater?
How many bacteria are in 1g of soil?
How many bacteria does out microbiome contain?
(How many different species)
- 1 million
- 40 million
500-100 different species
Name some of the differences in physical properties between bacteria
- Cell wall structure
- Staining characteristics
What do their genetic differences produce?
- Different metabolic features
- Different surface molecules
- Different antigenic properties
What are the smallest bacteria and how small are they?
What are the largest bacteria and how large are they?
What is the size of E.coli
]-The smallest are Archaea, such as Nanoarchaeum equitans --) 0.4 μm
]- The largest bacterium is the sulphur bacterium Thiomargarita namibiensis --) 750μm diameter
- E.coli is 1.8μm diameter and 7μm long
A genus of bacteria has one of three main shapes, and the shape is sometimes indicated in its name:
When is further differentiation possible?
]- Bacillus or rod-shaped, e.g Eschericha e.g Eschericha; Bacillus
]- Coccus or spherical, e.g Staphylococcus; Streptococcus
]- 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 pneumonia; in chains, e.g Streptococcus or in clusters, e.g. Staphylococcus;
Explain gram staining
What colour are Gram-positive bacteria after staining?
What colour are Gram-negative bacteria after staining?
- The gram stain allows microbiologists to distinguish between Gram positive and Gram negative bacteria
- The different staining properties are due to differences in the chemical composition of their cell walls
- Before staining, bacteria are colourless
- After staining, Gram-positive bacteria are stained violet
- After staining, Gram-negative bacteria are stained red
Explain the characteristics of bacterial cell walls
- The cell wall of all bacteria is a 3-dimentional network of polysaccharides and polypeptides, know as peptidoglycan or murein
- The cross-linking of these molecules provides strength and give the cell its 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
What are the four stages of Gram staining?
1. Crystal violet (BASIC DYE)
Binds to peptidoglycan so all bacteria stain purple
2. Lugol's iodine (MORDANT)
Binds the crystal violet to the peptidoglycan more strongly
3. Acetone-alcohol (DECOLORISER)
Removes unbound crystal violet and lipopolysaccharide:
]-Gram- negative bacteria lose their stain and become colourless
]- Gram positive bacteria remain purple
4. Safranin (COUNTER-STAIN)
]- Gram-negative bacteria stain red
]- Gram-positive bacteria remain purple
Explain Gram-positive bacteria with regard to staining
- 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 cell walls of Gram-positive bacteria allows them to bind stain efficiently and makes them more susceptible to the antibiotic, penicillin, and the enzyme, lysosome, than Gram-negative bacteria
- Bacteria constantly make 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 cells when they divide. Penicillin therefore makes the cell walls structurally weak and prone to collapse. Water uptake by osmosis bursts the cell
Explain Gram-negative bacteria with regard to staining
- Gram-negative bacteria have a more chemically complex cell wall than Gram-positive bacteria. Their outer membrane is supplemented with large molecules of lipopolysaccharide which protects the cell and exclude dyes like crystal violet, so they appear red or pink in the cell
- Gram-negative bacteria include Salmonella specie and E.coli. The outer lipopolysaccharide protects the peptidoglycan below and so they are not affected by lysosome and are resistant to penicillin. To control them requires a different class of antibiotics, that interfere with the cells 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
Conditions necessary for culturing bacteria
-Micro-organisms can undergo 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 substrate providing they are supplied with nutrients, water and suitable physical conditions, such as temperature
- Micro-organisms vary in their requirements and usually grow over a range of temperature and pH values with an optimum within the range
Micro-organisms require the following conditions for growth: Nutrients
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 carbon and energy source, usually glucose
- Nitrogen for amino acid synthesis, in organic molecules and in inorganic forms such as nitrate ions
Micro-organisms require the following conditions for growth: Temperature
AS bacterial metabolism is regulated by enzymes, the range of 25 -45°C is suitable for most bacteria. The optimum for mammalian pathogens is around 37° C the temperature of the human body
Micro-organisms require the following conditions for growth: pH
Most bacteria mare favoured by slightly alkaline conditions (pH 7.4), whereas fungi grow better in neutral to slightly acid conditions
Micro-organisms require the following conditions for growth: 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:
- A 'defined' medium contains only known ingredients
- An 'undefined' medium contains components that are not all known, because they include, for example, yeast extract or beef peptone
- 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
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
How do you prevent the contamination of pure cultures and apparatus by bacteria from the environment?
- Sterilise all apparatus and media before use to prevent initial contamination
- Handle cultures carefully, flaming the necks of the culture vessels before opening and closing and use equipment such as sterile loops to prevent subsequent contamination
How would you prevent contamination to the environment by the bacteria being used in experiments?
-] Sterilise the work surface before and after an experiment using a disinfectant, for example, 3% Lysol
-] 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:
why should cultures not be inoculated at 37°C
- 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
- FLAME the mouth of the bottle, for 2 or 3 seconds
- PASS the inoculating loop through a flame until it is red hot, and allow it to cool in the air
- LIFT the lid of the petri dish just enough to allow entry of the inoculating loop
- Secure the petri dish lid 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
- INCUBATE at around 25°C. Cultures should not be cultured at 37°C as this is an ideal temperature for the growth of many pathogenic micro-organisms
- DO NOT open petri dishes after incubation
how to properly dispose of materials?
After use disposable materials, such as plastic Petri dishes, can be sealed inside autoclavable plastic bags, autoclaved and then placed in a dustbin
Why is it important to estimate the growth of a population?
-Environment 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 grown in 200 dm3 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: DIRECTLY
By counting cells
-Viable counts describe living cells only
- Total counts describe living and dead cells
The size of a population of micro-organisms in liquid culture may be measured: INDIRECTLY
By measuring the turbidity (cloudiness) of the culture
- This method can be used as a field work technique, using light absorption by a sample of river water to indicate the number of bacteria present
Measuring growth directly: Planting and counting colonies
1. The sample is diluted using serial dilution. When 1 cm3 suspension is added to 9cm3 medium, it has been diluted 10 times and is a 10^-1 dilution
2. If this process is repeated, dilutions of 10^-2, 10^-3, 10^-4 and so on can be made
3. But if 0.1cm3 is progressively added to 9.9cm3, the first dilutions are 10^-4, 10^-6 and 10^-8
4. 1cm3 of each diluted sample is spread over sterile agar plate and incubated at 25°C for two days, allowing the bacteria to grow.
5. 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 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 =2.6 x 10^9
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