Microbiology Flashcards
How can medicines become contaminated?
- During manufacture: personnel, environment, raw materials (especially water), packaging
- During use by the patient
2 super kingdoms
Prokaryotes and eukaryotes
3 domains
bacteria, archaea, eukaryotes
Difference between prokaryotes and eukaryotes
- Prokaryotes are simple in their cellular organisation. Eukaryotes are much m
- ore complex.
The defining difference is the encapsulation of chromosomal DNA in the nucleus in eukaryotes, but there are many other differences too, particularly the presence of membrane-bound organelles such as mitochondria.
- ore complex.
Viruses
- Viruses are infectious particles made of genetic material (DNA or RNA) wrapped in a protein coat - they can only reproduce by hijacking the machinery of living cells and are not considered truly alive on their own.
Prions
Prions are even simpler than viruses, consisting only of misfolded proteins that can convert normal proteins to the same misfolded state. The most well-known prion disease is BSE (bovine spongiform encephalopathy) or “mad cow disease”, which can be transmitted to humans as variant Creutzfeldt-Jakob disease (vCJD)
Another example of a prion disease
scrapie, a prion disease that affects sheep and goats, which has been known to shepherds for centuries and helped scientists understand how these unique pathogens work.
Alzheimer’s, Parkinson’s, and Huntington’s
some researchers to investigate whether they might share some mechanisms with prion diseases.
What is visible and what isn’t?
- Single bacterial cells are not visible to the naked eye, but can be viewed under a light microscope if stained appropriately.
- Colonies of bacteria are visible to the naked eye. Each colony is formed of tens of billions of bacterial cells. Experienced microbiologists can use the appearance of a bacterial colony to help identify the organism.
Bacterial shape
2 common shapes: round and elongated
Round - cocci
(can grow as single cells, or as groups of bacteria together in clusters of chains. The particular way a bacteria grows can be further used to help identify it)
Elongated - rods
There is quite a lot of variation in shape between different bacteria which are rods. For example, some bacteria have a typical rod shape (see top image), some appear to be more rounded and appear more similar to a cocci (middle image), and some are curved rods (lower image). There is huge diversity amongst bacteria and thus other shapes are also known though are less common, such as spiral and filaments. The bacteria we will consider in these lectures will be predominantly cocci and rods.
Prokaryotic cell wall
- Confer cell shape
- Prevent the cell from bursting by providing rigidity
Cell wall is also the site of action of many important classes of antibiotics such as the penicillin or glycopeptides
- Prevent the cell from bursting by providing rigidity
Gram positive bacteria
- Take up Gram stain and appear purple under the microscope.
- Have a thick layer of peptidoglycan – a complex polymer which is heavily crosslinked.
Gram negative bacteria
- Do not retain Gram stain and appear red/pink under the microscope.
- Have a thin layer of peptidoglycan which is not as tightly crosslinked as that in Gram positive bacteria.
- Possess an additional outer membrane outside of the peptidoglycan layer which is not present in Gram positive bacteria.
Lipopolysaccharide (LPS) and the Gram-negative outer membrane (OM)
- Lipopolysaccharide (LPS) is a very important feature of the outer membrane in Gram negative bacteria. It is a complex molecule which is only found in the outer leaflet of the Gram negative outer membrane.
- The presence of LPS means that the outer membrane does not behave like a typical lipid membrane which we studied in PHAY0003.
- LPS is a potent regulator of the immune response, causing fever and sometimes causing septic shock.
- LPS is a huge problem in medicine sterilisation because it is extremely heat stable and therefore very difficult to remove during sterilisation processes.
Acid fast bacilli
- Gram-positive organisms with additional long chain ‘waxy’ mycolic acids in cell wall
- E.g. TB and leprosy
- Acid-fast bacteria are Gram positive, and therefore possess a thick layer of peptidoglycan in their cell wall. However, unlike other Gram positives, acidfast species possess a large amount of mycolic acids (a lipid with very long tails, C55) embedded in the outside surface of the cell wall. This makes the cell wall of acid-fast bacteria very hydrophobic and makes it very difficult for hydrophilic compounds to enter the cell.
Bacterial growth - how
- Binary fission
- Divide as fast as environmental conditions will allow (nutrients and temp)
Growth is exponential
- Divide as fast as environmental conditions will allow (nutrients and temp)
Bacterial growth curve
Lag phase
Log phase
Stationary phase
Death phase
Lag phase
Preparing to divide but have not yet undergone any division, so cell number remains the same
Log phase / exponential phase
Bacteria double at regular intervals and no grows exponentially - continues for as long as environmental conditions allow
Stationary phase
Cell death and cell growth are evenly matched, so there is no overall growth in the number of cells
Death phase
Number of alive cells begins to decrease
Why do cells enter the stationary phase?
Bacterial cells typically stop growing exponentially either because they run out of nutrients or because they become surrounded by toxic waste products which prevent their growth.
Many genes are upregulated at the end of log phase which help bacteria to survive when they are experiencing these harsh conditions. This is important because some of these genetic changes alter the bacterial cell properties, affecting how resistant they are to antibiotics and to the human body’s immune response.
Bacterial endospores
Bacillus and Clostridium species are two important examples of bacteria which undergo significant genetic changes as they go into stationary phase.
These are the only two types of bacteria which form endospores – a change of bacterial morphology which helps the cell to survive during stationary phase.
Forming endospores enables these bacteria to survive harsh environmental conditions (e.g. lack of nutrients or water, adverse pH or temperature) and makes them resistant to chemical disinfection. From a sterilisation perspective, this makes them very difficult to get rid of!
Endospores for Bacillus species are therefore used as biological indicators in sterilisation processes. Once conditions become favourable, endospores can germinate, allowing the bacteria to grow.
Calculating population size during log phase
Look at notes
Measurement of cell numbers
- Counting the number of cells using a microscope. This is not always the best method because it cannot distinguish between viable (i.e. alive) cells and dead ones.
- Viable counting. This is the method which is most commonly used. In this method an agar plate is spread with a solution containing bacteria. The plate is incubated for 24 hours and then the number of colonies is counted. Each colony arises from a single bacterial cell which has undergone multiple rounds of doubling, and therefore each colony present on the plate represents 1 single bacterium which was present to begin with.
Bacterial growth requirements
- Culture media
- Oxygen requirement
- Temperature
Culture media types
Defined media
Complex media
Selective media
Differential media
Defined media
contains known quantities of all the different components in the media. These types of media are important for research.
Complex media
Often used for routine microbiology and are typically meat or yeast extracts. These media can be liquid (for growing bacteria in suspension) or can be made solid by addition of agar (for growing bacteria in Petri dishes or well plates).
Selective media
favours the growth of particular organisms over others which can be useful for identification.
Differential media
can also be used to help with identify the type of bacteria which is growing. A common example is media containing a pH indicator.
Oxygen requirements
Oligate aerobes
Facultative anaerobes
Obligate anaerobes
Obligate aerobes
The presence of oxygen is obligatory for growth of these bacteria, because their metabolism requires oxygen.
Facultative anaerobes
These bacteria can survive either with or without oxygen. Depending on whether oxygen is available, the bacteria are able to change their metabolism.
Obligate anaerobes
These bacteria are completely inhibited by the presence of oxygen and will not grow at all if oxygen is present.
How do we take special measures to enable growth of obligate anaerobes in a lab?
- Oxygen can be physically excluded from the growth environment. Previously this was done with equipment called an anaerobic jar, but now can be done with specialised equipment which maintains atmospheres with no oxygen.
- Oxygen can also be chemically reduced or removed from culture media, usually by the addition of a reagent which will react with oxygen.
Temperature exceptiosn
Thermophiles and psychrophiles
Thermophiles
These organisms grow best in hot conditions, some can even grow happily in temperatures up to ~100 °C. Thermophiles are often found naturally in extreme environments, such as in hot springs.
Psychrophiles
These organisms grow in low temperatures, even in the fridge. This has important implications for medicine storage.
Identification of unknown bacteria (5)
- Colony morphology
- Selective media
- Gram staining
- Oxygen requirement
- Biochemical tests
Colony morphology
In other words what does the colony look like
Selective media
Growing the bacteria on selective media can provide clues about which conditions the organism needs to be able to grow
Gram staining
Always routinely conducted to determine whether the organism is gram+ or gram-. bacteria will also be examined under a light microscope, so the shape of the bacterial cell can also be determined
Oxygen requirement
Growing the organism in air will determine whether the organism is an aerobe or obligate anaerobe
Biochemical tests
A range of simple tests can be conducted to determine exactly which species is present
Staphylococcus aureus (Gram +)
- Very common nosocomial (hopital acquireD) pathogen
- Huge range of infections: superficial skin infections, UTI, septicaemia, endocarditis
What might presence indicate?
Personnel involved in medicine preparation are possible shedders
A small minority of people carry these organisms and are chronic shedders. They should not be involved with food / pharmaceuticals productions
Why is S aureas problematic
Very common, including in clinical settings
Can cause serious infection
Other important Gram+ organisms
Bacillus sp.
Clostridium sp.
Mycobacterium sp.
Strepococcus sp.
Escherichia coli (Gram -)
Small component of normal bowel flora
Large range of infections: UTI, gastrointestinal
What might presence indicate?
Problems with water quality and suggestive of faecal contamination of water supply
Pseudomonas aeruginosa (Gram-)
Can survive and multiply in simple aqueous environments e.g. sinks / showerheads / aqeuous pharmaceuticals
Very resistant to many antimicrobials
Cause of very serious infections:
* Serious eye infections and would / chest infections, particularly in burn patients
What might presence indicate?
Problems with water systems
Other important Gram- organisms
Bordetella pertussis (whoopingcough)
Haemophilus influenzae (meningitis)
Legionella pneumophila (Legionnaires’ disease)
Neisseria sp. (Bacterial meningitis, N. gonorrhoea)
Salmonella sp.
Vibrio sp.
