Microbiology (ICS) Flashcards
What is the process of gram staining?
1) Stain the slide with CRYSTAL VIOLET (30-40seconds) then rinse with water.
2) Stain it with iodine (1 min)
3) Wash the slide with Acetate/Alcohol (slide is tilted, and alcohol dripped using dropper) –> At this stage, gram positive will be purple and gram negative bacteria will be colourless
4) Use safranin counterstain (20-30sec) –> Gram positive bacteria will be purple and gram negative bacteria will be pink.
What is a commensal?
What is an opportunist pathogen?
An organism which colonises the host but causes no disease in normal circumstance. (occasionally they can cause infections, especially when the patient is immunocompromised)
Opportunist pathogen - a microbe which only causes disease if host defences are compromised.
What are the differences in structure between gram positive and gram negative bacteria?
Explain using this why the gram positive bacteria retain the crystal violet-iodine stain and gram negative bacteria do not.
Gram positive bacteria
- Thick peptidoglycan layer
- Lack an outer membrane
- Have lipoteichoic acid which provides structural support
Gram negative bacteria
- Thin peptidoglycan layer
- Have an outer membrane composed of lipopolysaccharides (endotoxin)
The thick peptidoglycan layer of gram positive bacteria have extensive cross-linking of peptide chains which form a dense meshwork to trap the crystal violet-iodine complex.
The presence of the lipid rich outer membrane in gram negative bacteria is not permeable to the crystal violet-iodine complex. - so alcohol/acetate washes off the stain
What is an obligate? e.g. obligate intracellular bacteria
An organism. which requires a host cell in order to survive and replicate.
What is endotoxin and exotoxin?
Endotoxin - Component of the outer membrane in bacteria e.g. lipopolysaccharide in gram negative bacteria
Exotoxin - Secreted proteins of both gram positive and negative bacteria.
What are the differences between endotoxin and exotoxin?
Exotoxin - composed of protein
Endotoxin - composed of lipopolysaccharide
Action
Exotoxin - Specific (target specific cell types, specific receptors)
Endotoxin - Non-specific (generalised effect)
Heat sensitivity:
Exotoxin - Heat labile and can be neutralised by heat
Endotoxin - Heat stable, not easily neutralised by heat
Exotoxin - Produced by both gram positive and negative bacteria
Endotoxin - Found only in gram negative bacteria.
What are the methods of genetic variation in bacteria?
Mutation - Base substitution, deletion, insertion
Gene transfer
1) Transformation via UPTAKE of free DNA from the environment
2) Transduction via phage - DNA is transferred from one bacterium to another by a virus.
3) Conjugation via sex pilus - Bacterium transfers genetic material to another through direct contact. ( via a plasmid e.g. transfer promotion genes, plasmid maintenance genes, antibiotic or virulence determinant genes)
What stain do you use for mycobacteria? Why?
Ziehl-Neelsen stain. This is because their cell wall has a high lipid content with mycolic acids, which makes them resistant to gram stain .
Their cell wall contains lipoarabinomannan.
What are some challenges when dealing with mycobacteria e.g. M.tuberculosis?
1) It has a thick lipid rich cell wall making it difficult for immune cells to effectively kill it and for drugs to penetrate to exert their effect.
2) They have a slow growth rate, meaning that symptoms would gradually develop over time, which leads to a delay in diagnosis and treatment.
What are the 3 main forms of tuberculosis?
Primary tuberculosis
- Occurs when a person is first exposed to M.tuberculosis.
- Although initially alveolar macrophages can kill the bacteria, some bacteria survive within the macrophage and spread to the hilar lymph nodes (within the macrophage, within the lymphatic system) where immune responses are triggered.
Granuloma + lymphatics + lymph nodes = primary complex.
Latent tuberculosis
- Individuals do not exhibit symptoms as bacteria are in a dormant state.
- T cells specific to M.tuberculosis antigen are still active to maintain the immune response.
- Latent TB is detectable on a tuberculin skin test
Pulmonary tuberculosis
- Granulomas form around the bacilli (usually in the apex of the lungs) to control the infection
- Some granulomas can undergo caeseous necrosis which lead to formation of abscesses)
- TB can spread within the lungs leading to additional lesion and abscess formation
Take note: Pulmonary TB can occur immediately following a primary infection or months later following a reactivation of latent TB
Describe Blood, Chocolate and Cled Agar
Blood - Contains Sheep or horse blood and provides a good medium for growing many different types of bacteria
Chocolate - Contains blood agar heated to 80degrees celcius for 5 mins to release some nutrients which make it easier for FASTIDIOUS organisms to grow - an organism which has a complex or particular nutritional requirement)
Cled- Cysteine lactose electrolyte deficient –> Used to differentiate micro-organisms in urine. You can identify lactose fermenting bacteria (E.coli) as yellow and non-lactose fermenting bacteria (Salmonella, shigella) as blue.
What are drug resistance mechanisms that mycobacteria have?
- Drug inactivation via production of beta-lactamase
- Target overproduction – bacteria increase production of another target which is specific for an enzyme or protein of the drug which makes it difficult for the drug to exert its effect.
- Alteration of drug target - Missense mutations can modify the structure of target proteins which reduces the drug’s affinity for the bacterial antigen.
- Altered cell envelope - mycobacteria can modify its cell envelope to increase permeability and drug efflux.
Take note: XDR-TB (resistant to the 4 commonly used TB drugs) is very complicated to treat and that treatment can fail with TTR TB (totally drug resistant tuberculosis)
Describe MacConkey, XLD and Gonococcus agar.
State the colour of any differentiating agar
MacConkey - Primarily grows gram negative bacilli –> Allow for differentiation between lactose fermenting bacteria (E.coli)- as pink and non-lactose fermenting bacteria (Salmonella, shigella)- as yellow/colourless.
XLD - Xylose lysine deoxycholate - Very selective growth medium used to differentiate/isolate salmonella species and shigella species. (Red at pH 7.4)
Most gut bacteria ferment xylose and lower the pH turning the colour yellow.
Shigella –> Red colonies
Salmonella –> Red with black centres (salmonella reduces thiosulphate to hydrogen sulphide)
Gonococcus agar - Used for Neisseria cultures. (Neisseria species)
Describe Sabourad’s agar, CCDA agar and Lowenstein Jensen medium
Sabourad’s agar - Used to culture fungi. (Inhibition of bacteria is aided by presence of antibiotics)
CCDA agar - Charcoal cefoperazone deoxycholate agar. –> Selective and supports growth of Campylobacter
Lowenstein Jensen medium - Used for mycobacterium species. e.g. M.tuberculosis.
Draw the gram positive cocci map
Check with sheet
Draw the gram negative cocci map
Check with sheet
What is lancefield type grouping and what types of diseases is each group of antibiotics likely to cause?
The lancefield test uses antibodies (bound to latex beads) to detect antigens on the surface of beta haemolytic streptococci. (determined based on clumping of specific antibody e.g. group A,B,C to antigen) so if bacteria do not have the corresponding antigen to antibodies, clumping doesn’t occur.
A,C,G: Tonsilitis and skin infection
B: Neonatal sepsis and meningitis
D: Urinary tract infection = enterococci.
Which are the sterile sites of the body?
If there happen to be active bacteria, what could it cause in each of the sites?
Blood - Sepsis
CSF - Meningitis
Pleural fluid - Pericarditis, pleural effusion
Peritoneal cavity - spontaneous bacterial peritonitis
Joints - Septic arthritis
Urinary tract - UTIs
Lower respiratory tract - TB + pneumonia
Where are the common sites of bacterial colonies?
Mouth - alpha haemolytic strep, corynebacterium species, non-pathogenic neisseria species, non-pathogenic haemophilus species
Skin- Staphylococcus epidermidis, corynebacterium species, proprionibacterium acnues, enteric bacilli
Vagina - Streptococcus species, bacteroides species, lactobacillus acidophilus (which produces lactic acid keeping vaginal secretions acidic to prevent pathogenic bacteria from establishing infection)
Urethra - Enterobacteriacea, staphylococcus saprophyticus
Large intestine:
Anaerobes - Clostridium species and bacteroides species
Aerobes - enterococci, lactobacilli, E.coli
What tests could you do to diagnose viral infections?
- Electron microscopy (takes a long time and is operator dependent but it can detect any virus even a novel one)
- PCR - fast and sensitive but there is a risk of false positive. (Also, not possible to detect a virus unless you suspect it first and use relevant primers) –. diagnostic swab would be a green viral swab (whereas black charcoal swab for bacteria)
- Serology - Detection of antibody responses in serum - can indicate current or past infection/vaccination
ELISA - Enzyme linked immunosorbent assay - detects antibodies using an enzyme based reaction.
What is a purple and yellow top tube for? How long will results take to get back for each?
Purple top tube - FBC and film. - to detect atypical lymphocytes - results should be out on the same day
Yellow top tube - Serum - detects antibodies to virus - results take 1-2 days to come out.
What is a main differential for EBV? How would you diagnose EBV?
An infection caused by S.pyogenes.
They both present with tonsillitis with a purulent (discharging pus) lining of the tonsils.
To differentiate you would do a blood test (FBC) and check for atypical lymphocytes (irregular shape and larger nucleus)
OR
do a serology test to check for IgM antibodies for EBV - acute infection. IgG antibodies for EBV- chronic/prior infection.
(In some cases can check for antistreptolysin which is an antibody for S.pyogenes as it produces streptolysin toxin)
Examples of vaccine preventable diseases
Diphtheria, poliomyelitis, tetanus, hepatitis B, pertussis, meningitis, haemophilus influenzae type B
What is a virus?
What is a virion?
An infectious, obligate intracellular parasite which comprises genetic material surrounded by a protein coat and/or membrane.
A virion is the infectious particle of a virus which exists outside host cells. It contains genetic material and has an outer protein shell - capdis (A virus encompasses both the virion and intracellular components)
Differences between virus and bacteria
Virus - No cell wall
Bacteria - have cell wall
Virus - no organelles
Bacteria- have organelles
Virus - Dependent on the host cell
Bacteria - not dependent on the host cell
Virus - Do not have both DNA and RNA (only 1 of them)
Bacteria - Has both DNA and RNA
What is the process of viral replication?
1) The virus attaches to a specific receptor on a cell
2) (Only) The virion (viral core) enters the cell and undergoes uncoating (removal of protein coat to expose viral genetic material)
3) The viral genome migrates to the cell nucleus and it is transcribed to mRNA using host materials (Enzymes, nucleotides, amino acids)
4) The viral mRNA is translated to produce structural proteins, viral genome and non-structural proteins e.g. enzymes
5) The virion is assembled, which can occur in the nucleus (herpes virus), cytoplasm (poliovirus) or at the cell membrane (influenza virus)
6) The new virus particles are released either via cell lysis or bursting:
= The cell ruptures, releasing accumulated viral particles into the extracellular space
= The assembled virion bud out of the host’s cell membrane, exiting the cell while acquiring an envelope (lipid coat) from the host’s cell membrane.
–> In lysis, the cell dies.
–> In budding, the host cell does not die immediately and can continue to produce more virus.
How do viruses cause disease?
1) Direct destruction of host cells –> poliovirus causes host cell lysis and death after viral replication (of 4 hours) –> paralysis
2) Modification of host cell –> rotavirus can cause atrophy of villi and flatten epithelial cells – decreasing the small intestine surface area for absorption of nutrients – leading to a hyperosmotic state (low concentration of solute) – PROFUSE diarrhoea.
3) Over-reactivity of the immune system –> Hep B can cause jaundice, pale stool, dark urine, etc.
- Hep B triggers activation of the cytotoxic T cells which lyse the whole host cell (as they can’t kill only the virus).
- In some cases the immune system produces excessive cytokines which can lead to severe inflammation, tissue damage and organ failure (cytokine storm)
4) Damage through cell proliferation –> HPV acquired through sexual contact - viral DNA is integrated into host chromosome and every time the host DNA is replicated, more viral proteins are made which interact with tumour suppressor genes of the cell –> leading to abnormal cell proliferation (carcinoma in situ)
5) Evasion of host defence
- Latency - when a virus becomes dormant and will reactivate in the future
- Antigenic variability due to small mutations over time which accumulate to change the antigen.
- It interferes with the host cell antigen processing pathway
- Down regulation of interferon and other intracellular host defence proteins.
How does a virus evade host defence?
Latency - when a virus becomes dormant and will reactivate in the future
- Antigenic variability due to small mutations over time which accumulate to change the antigen.
- It interferes with the host cell antigen processing pathway
- Down regulation of interferon and other intracellular host defence proteins.
What is meningitis? What are its infectious and non-infectious causes?
Meningitis is the inflammation of the meninges. (commonly affects extremes of ages due to impaired/waning immunity)
Main bacterial causes - S.pneumoniae (pneumococcus), N.meningitidis (meningococcus).
Main viral causes - Coxsackievirus, herpes virus
(Rarely caused by fungi and protozoa)
Non infectious causes
- Medication - antibiotics, NSAIDS
- Cancer - melanoma, lung cancer, lymphoma, leukemia
- Autoimmune disease - SLE, Behcet’s syndrome
What do you do when you encounter a notifiable disease?
How will this protect the community?
(It is a legal obligation for any doctor that suspects a case to report on clinical suspicious)
Doctors should notify UKHSA - UK Health Security Agency ASAP within 3 days of a case being notified or within 24 hours for urgent cases.
This will protect the community by:
- Investigation- Contact tracing, partner notification
- Identify and protect vulnerable people e.g. by chemoprophylaxis, immunisation, isolation
- Educate, inform, raise awareness
