4.4 Microbiology

Standard metric units

Prokaryotic Cells Revision: What’s a prokaryote? What are the key features of prokaryotes?

Capsule Cell wall Ribosomes Nucleoid Flagella Pili / fimbriae Cytoplasm

Cell wall Thick outer covering that maintains the overall shape of the bacterial cell

Ribosomes cell part where proteins are made Ribosomes give the cytoplasm of bacteria a granular appearance in electron micrographs

Nucleoid a ring of DNA

Flagellum a whip-like “tail” that some bacteria have. Used for locomotion

Fimbriae hollow hair-like structures made of protein allows bacteria to attach to other cells or surfaces Fimbria - singular Fimbriae - plural

Cytoplasm clear jelly-like material that makes up most of the cell

So why are they this shape? Bacterial Shapes So why are they this shape?

Functions of the bacterial cell wall Maintain the shape of bacterium Rigid - protecting cell from osmotic lysis Exchange nutrients and chemicals Antigenicity Related to its pathogenesis

Gram’s stain To differentiate between Gram positive and negative bacteria

Growing bacteria in the lab Bacteria can be grown if given the right conditions, nutrients and water. There are a number of physical factors that effect bacterial growth: Temperature pH Oxygen availability

Physical Factors for bacterial growth Temperature Psychrophiles grow optimally below 15°C Thermophiles multiply best around 60°C Hyperthermophiles are Archaea that grow optimally above 80°C Mesophiles thrive at the medium temperature range of 10° to 45°C, including pathogens that thrive in the human body

Most bacteria are happiest at 37oC and wont grow much about 42oC Some can produce spores and survive at very high temperatures e.g. Bacillus Spores can resist sterilisation and can be used as an indicator

Oxygen obligate aerobes require oxygen to grow Anaerobes do not or cannot use oxygen Facultative anaerobes grow either with oxygen or in reduced oxygen environments

pH The majority of species grow optimally at neutral (~7.0) pH Acidophiles are acid-tolerant prokaryotes For example, those used to turn milk into buttermilk, sour cream, and yogurt

These are supplied in a nutrient media Nutrients These are supplied in a nutrient media Carbon – usually in an organic form eg glucose Nitrogen – organic or inorganic Other growth factors – vitamins and minerals The usual source of energy used is glucose

Factors affecting bacterial growth All of these factors can affect bacterial growth: Extend lag phase Decrease exponential (log) phase Premature stationary/death phase

Bacterial growth curve

The ‘perfect’ growth curve Can be used to calculate growth rate Can be used to calculate generation time Need linear part of growth curve Calculate growth rate first Calculate generation time once you know the growth rate

Generation times Linear part Growth rate (k) = log10Xt – log10X0 T  Generation time = 1/(k)

arithmetic semi-log Rate of growth Choose two points on linear part of graph Higher value is Xt Lower value is X0 Measure time interval between them (T) Log the Xt and X0 values and put into following formula: T Gives gen/hr (k) Calculate generation time: gen time = 1/k Gives answer in hr per gen convert to min/gen by multiplying the answer by 60 arithmetic semilog

Handling cultures in the lab

Aseptic technique Avoid contamination!

Sterile equipment Equipment and media must be sterilised Autoclave

Sterile equipment Equipment and media must be sterilised Autoclave 121˚C for 15mins Under pressure Bunsen for inoculating loops Heat labile plastics are irradiated Must be protected from contamination after sterilisation

Methods for measurement of cell numbers Direct microscopic counts are possible using special slides known as a haemocytometer. Dead cells cannot be distinguished from living ones. Only dense suspensions can be counted (>107 cells per ml), but samples can be concentrated by centrifugation or filtration to increase sensitivity.

Indirect viable cell counts, also called plate counts, involve plating out (spreading) a sample of a culture on a nutrient agar surface. The sample or cell suspension can be diluted in a nontoxic diluent (e.g. water or saline) before plating.  If plated on a suitable medium, each viable unit grows and forms a colony. Each colony that can be counted is called a colony forming unit (cfu) and the number of cfu's is related to the viable number of bacteria in the sample.

Estimating size of viable bacterial population 4.5 ml 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8

1 ml 10-4 10-5 10-6 10-7 10-8 Gently swirl plate to mix

Make sure you work close to the bunsen burner Pouring an agar plate Make sure you work close to the bunsen burner Flame the neck of the bottle

Motile bacteria Some bacteria are motile: These tend to be Gram negative rods Flagellar make bacteria motile These can be stained Unstained bacteria can be visualised swimming using light microscopy

Motility testing The hanging drop method: Bacteria are suspended in a drop of liquid They can be seen by light microscopy Motile bacteria swim in straight line Non-motile bacteria ‘vibrate’ a bit (Brownian motion)

Motile bacteria Non-motile bacteria (Brownian motion)

Using bacteria commercially A lot of commercial activities use microbiology directly in their processes Food manufacturing (think yogurt, cheese, tofu and brewing) Drug production Waster water treatment, bioremediation etc It is truly applied biology!

Streak plate to obtain a pure culture Many processes require pure cultures to start with. How do you do that in the lab? Streak Plates!

The microorganisms are grown in very large vessels called fermenters  

The large stainless steel cavity is filled with a sterile nutrient solution, which is then inoculated with a pure culture of the carefully selected fungus or bacterium. Paddles rotate the mixture so that the suspension is mixed well. As the nutrients are used up, more can be added. Probes monitor the mixture and changes in pH, oxygen concentration and temperature are all computer controlled.

A water jacket surrounding the fermenter contains fast flowing cold water to cool the fermenter since fermentation is a heat generating process. Most of the air, including carbon dioxide and other gases produced by cell metabolism, leave the fermenter by an exhaust pipe. There are two main types of culture used in industrial processes: batch cultures and continuous cultures.

Batch Culture Continuous Culture cells are grown in a fixed volume of liquid medium in a closed vessel nutrients are added and cells harvested at a constant rate No microorganisms, fluid or nutrients are added or removed from the culture during the incubation period Volume of suspension is kept constant Used for producing secondary metabolites, such as penicillin and other antibiotics, which are relatively unstable and not essential for the growth of the culture Fermenter does not have to be emptied, cleaned and refilled very often Secondary metabolites can be extracted economically only when they reach a high concentration in the culture Production is almost continuous Continuous cultivation needs sophisticated equipment to maintain constant conditions.  Highly trained staff need to operate the equipment.  Therefore this process can be expensive

Penicillin Production Penicillium notatum (fungus) grown in batch culture It produces the antibiotic after the growth (log) phase when glucose is depleted (limiting) Can you think why it does that? Made to reduce competition for its food The fungal mycelium by filtration The residual liquid is then processed to purify the antibiotic.

For more info on penicillin production: http://penicillin. wikispaces