Bio 3.4 - Microbiology

How can bacteria be distinguished from each other?

• size

• shape

• staining characteristics

• metabolic features

• antigenic features

• genetic features

What are the three shapes of bacteria?

• Bacillus

• Coccus

• Spirillum 

What shape is bacillus?

Rod shaped

What shape is coccus?

Spherical

What shape is spirillum?

Spiral/corkscrew 

How are bacteria grouped?

• singly

• in pairs

• in chains

• in clusters 

Using cell structure, what are the two types of bacteria?

• Gram positive

• Gram negative 

How do you distinguish between gram positive and negative bacteria? 

Using gram stain 

What colour does gram positive bacteria stain after using gram stain?

purple

What colour does gram negative bacteria stain after using gram stain?

red

What are the 4 stages of gram staining?

• crystal violet (purple) - stain/dye

• Lugol’s iodine - mordant

• Ethanol - decolouriser

• Safranin (red) - counter stain 

What is the effect of the reagent crystal violet?

Binds to the peptidoglycan cell

• all cells will appear purple 

What is the effect of the reagent Lugol’s iodine?

Helps bind the crystal violet to peptidoglycan cell wall strongly

What is the effect of the reagent ethanol?

Removes any unbound crystal violet stain and also the outer lipopolysaccharide layer

• gram positive = purple

• gram negative = colourless

What is the effect of the reagent safranin?

Binds to the peptidoglycan wall 

• gram positive = purple

• gram negative = red 

Explain the composition of the cell wall in gram positive bacteria linking it to why they stain purple?

• They retain the crystal violet/iodine complex

• Walls have a thick layer of peptidoglycan/murein

• Gram stain can easily reach it

• Peptidoglycan layer retains the crystal violet/iodine complex when washed with alcohol

• Do not have an additional lipopolysaccharide layer or protein layer

Why are gram positive bacteria susceptible to antibiotics such as penicillin?

• Penicillin inhibits the formation of cross linkages in the peptidoglycan layer which provides strength 

• weakens the wall and when water enters by osmosis the cell swells and bursts

• especially significant when cells divide 

Explain the composition of the cell wall in gram negative bacteria linking it to why they stain red?

• Have an additional lipopolysaccharide and protein layer outside the thin peptidoglycan layer

• This outer membrane reduces the penetration of the crystal violet stain 

• On treatment w alcohol the outer layer is lost and thin inner peptidoglycan layer is exposed

• Crystal/iodine complex is washed away 

• Bacteria show counterstain safranin which is red 

Why are gram negative bacteria not susceptible to antibiotics such as penicillin?

• Thick lipopolysaccharide layer prevents the entry of those molecules

• different antibiotics are required

Why do antibiotics not damage human cells as well as bacteria cells?

• Human cells do not have a peptidoglycan cell wall

• Human cells have 80s ribosomes not 70s - have a different metabolism to bacteria

What conditions need to be considered for culturing bacteria?

• Nutrients

• Growth factors

• Temperature

• pH

• Oxygen

What nutrients are needed for culturing bacteria?

• Supplied in a nutrient media

• Cultured in liquid medium - nutrient broth/medium solidified with agar

• Provides water 

Includes

• carbon - energy source (glucose)

• nitrogen for amino acids synthesis in organic molecules and in inorganic form e.g. nitrate ions 

What growth factors are needed for culturing bacteria?

Vitamins

• biotin

• mineral salts e.g. Na⁺, Mg²⁺, Cl^-, SO₄^2-, PO₄³-

• amino acids 

• purines + pyrimidines 

What temperature is needed for culturing bacteria?

• Bacterial metabolism is regulated by enzymes

• range of 25-45 C is suitable for most bacteria

• optimum for mammalian pathogens is 37

• suitable for enzyme activity 

What pH is needed for culturing bacteria?

• favoured by slightly alkaline conditions (pH 7.4) 

• fungi grow better in neutral to slightly acidic conditions

What is an obligate aerobes?

must have oxygen available

What are obligate anaerobes?

must not have oxygen

What are facultative anaerobes

can grow with or without oxygen but better with oxygen present

What is defined media?

contains only known ingredients/known concentrations

What is undefined media?

not all ingredients/concentrations are known e.g. yeast extract

What is selective media?

allows only certain bacteria to grow e.g. Maconkey agar - allows gram negative agar

What is the advantage of adding glucose rather than lactose to bacteria growing in a fermenter?

• All bacteria can ferment/utilise glucose for respiration

Two types of bacteria

• lactose - fermenting bacteria - can use lactose

• non lactose fermenting bacteria - can’t use lactose - can’t synthesise enzyme needed to hydrolyse lactose into galactose and glucose

What is aseptic technique?

Laboratory practice that maintains sterility in apparatus and prevents contamination of the equipment and the environment 

How do you prevent 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 necks of culture vessels before opening and closing

• Use equipment such as sterile loops to prevent subsequent contamination 

How do you prevent contamination by the bacteria being used in experiments? 

• Sterilise the work surface before and after an experiment using a disinfectant (1% virkon)

• Use the correct handling techniques to prevent the contamination of personnel and the immediate environment by the organisms being cultured

Why are petri dishes and nutrient agar sterilised before agar is poured? 

To prevent contamination of the culture medium 

How is an inoculating loop sterilised?

By heating it to red heat in a bunsen burner blue flame before and after use 

Why should the petri dish lid only be slightly lifted?

To prevent microorganisms from the air contaminating the culture and vice versa 

How do you secure a petri dish after inoculation?

Lid should be secured with adhesive tape and label it with date and name

Why are inoculated agar plates incubated at 25 degrees for 24-48 hours in schools?

It encourages the growth of the culture without growing harmful or toxic pathogens 

What is an autoclave?

A large sealed container like a pressure cooker

What is an autoclave used for?

To sterilise cultures or glass and metal

• 121 degrees

• 15 mins

• in steam under pressure 

How do you dispose of petri dishes after use?

• Seal them in an autoclave bag

• autoclave at 121 degrees for 15-30 mins

• throw them out with general waste 

What sterilisation method is used for heat-labile plastics?

Irradiation

When might estimating bacterial population size may be important?

• Tissue samples e.g. urine 

• Water sample 

• Food samples/drug manufacture e.g. penicillin

What are the two ways the population size of bacteria in liquid culture may be measured? 

• Directly - counting cells 

• Indirectly - by measuring turbidity (cloudiness) 

What are the two types of direct counts when measuring the population size of bacteria in a liquid?

• Viable counts 

• Total counts

What is a viable count of bacteria?

Describes living cells only

What is a total count of bacteria?

Describes living and dead cells

How is the bacterial growth measured indirectly?

By measuring the turbidity of the culture - light absorption by sample of water gives an indication of the population of bacteria