ICS - Microbiology Flashcards
What does gram staining do?
Differentiate between gram positive and gram negative bacteria
Describe the gram staining process
- Fixation to microscope slide (heat/methanol)
- Primary stain - crystal violet
- Mordant - iodine
- Decolourisation - ethanol or acetone
- Counterstain - safranin
What colours does gram staining turn gram positive and negative bacteria?
Gram positive - purple
Gram negative - pink
What bacteria does not stain with gram staining?
Acid fast bacilli, mainly mycobacteria (e.g. TB)
Why do mycobacteria not stain with gram staining?
Have a waxy, lipid cell wall that is impenetrable to the gram stain (also allows them to withstand phagolysosomal killing)
What stain is used for mycobacteria?
Ziehl-Neelsen stain
Describe the Ziehl-Neelsen stain process
Use heat to drive a dye (carbon fuchsin) inside the cell wall
What colours does Ziehl-Neelsen staining turn bacteria?
Acid fast bacteria (positive mycobacterium) - red
Non-acid fast bacteria - blue
Define pathogen
Organism that causes or is capable of causing disease
Define commensal
Organism which colonises the host but causes no disease in normal circumstances
Define opportunist pathogen
Microbe that only causes disease if host defences are compromised
Define virulence/pathogenicity
How easily a pathogen is spread
E.g. high virulence = easily spread e.g. measles
Define asymptomatic carriage
When a pathogen is carried harmlessly at a tissue site where it causes no disease
Describe cocci
Round-shaped bacteria. Can be individual, as pairs, (diplococci), in chains or clusters
Describe bacilli
Rod-shaped bacteria. Can be individual, chains, vibrio (curved), spirochaete (spiral)
Describe blood agar
- Contains sheep/horse blood
- Provides a good medium for growing many different types of bacteria (non selective)
Describe chocolate agar
- Blood agar heated to 80 degrees celsius for 5 minutes
- Releases nutrients into agar and makes it easier to grow certain organisms (fastidious bacteria)
What are 2 examples of bacterias that grow better on chocolate agar?
- Haemophilus influenza
- Neisseria meningitidis
Define fastidious bacteria
Bacteria that is hard to grow because they have complex/restricted nutritional and/or environmental requirements
Describe MacConkey agar
- Contains bile salts (inhibits gram positive), lactose and pH indicator
- Designed primarily to grow gram negative bacilli
- Enables differentiation of gram negative bacilli into lactose fermenters and non-lactose fermenters
What colour are gram negative bacilli on MacConkey agar?
Lactose fermenting - red
Non-lactose fermenting - white/colourless
Describe CLED agar
- Cysteine lactose electrolyte deficient
- Use to differentiate microorganisms in urine
- Enables differentiation of lactose fermenting and non lactose fermenting gram negative bacilli
What colour are gram negative bacilli on CLED agar?
Lactose fermenting - yellow
Non lactose fermenting - blue
Describe Sabouraud’s agar
- For fungi
- Selective media
Describe XLD agar
- Xylose lysine deoxycholate agar
- Selective media
- Contains indicator
Describe Lowenstein-Jensen agar
- Specifically used for culture of mycobacterium species
- Selective media
Which sites are sterile in the body?
- Blood
- CSF
- Joints
- Lower resp tract
- Pleural fluid
- Peroneal cavity
- Urinary tract
Which sites are colonised with flora in the body?
- GI tract
- Skin
- Oral cavity (upper resp tract)
- Vagina
- Urethra
- Bladder
What is alpha beta gamma haemolysis?
Used to indicate different types of streptococci on blood agar plates
What test is used to differentiate beta haemolytic streptococci?
Streptoccoci Lancefield grouping test
What are the results of streptococcus lancefield grouping test?
Group A = strep. pyogenes
Group B = strep. agalactiae
Group C = strep. dysgalactiae
Group D = enterococcus
Describe the results of alpha beta gamma haemolysis
- Alpha - green/brown
- Beta - clear
- Gamma - neither (may be slightly discolouration in the medium)
What is the optochin test?
- Used in the identification of streptococcus pneumoniae
- Filter paper discs are impregnated with ethylhydrocupreine hydrochloride and applied directly to inoculated plates before incubation
Describe the results of the optochin test
- If a culture is resistant to optochin, the disc won’t prevent cultures growing
- If a culture is sensitive (susceptible) to optochin, the disc prevents cultures growing (streptococcus pneumoniae)
What is the coagulase test?
Used to differentiate bacteria that produce coagulase (e.g. staphylococcus aureus) from those that do not (e.g staphylococcus epidermidis/saprophyticus)
What is coagulase?
Enzyme-like protein that causes plasma to clot by converting fibrinogen to fibrin
Describe the results of the coagulase test
If coagulase is present, clotting will occur
What is the catalase test?
Used to differentiate bacteria that produce catalase (e.g. staphylococci) from those that do not (e.g streptococci)
What is catalase?
Enzyme that catalyses the release of oxygen from hydrogen peroxide
Describe the results of the catalase test
Hydrogen peroxide (H2O2) is added and if catalase is present, H2O2 will be broken down into 2H2O + O2 (gas bubbles)
Describe the characteristic features of gram positive bacteria
Single membrane and thick peptidoglycan layer
Describe the characteristic features of gram negative bacteria
Double membrane, thin peptidoglycan layer and lipopolysaccharides (endotoxin area)
What does the endotoxin area of gram negative bacteria comprise?
Terminal sugars. O antigen and Lipid A
Between what temperatures and pH range can bacteria grow?
-80 to +80 degrees celsius and pH of 4-9
What are the 3 phases of bacterial growth?
- Lag phase
- Exponential phase
- Stationary phase
Give an example of slow growing bacteria
TB
Give 2 examples of fast growing bacteria
E.coli and Staphylococcus. aureus
Give 2 functions of pili
- Help adhere to cell surfaces
- Plasmid exchange
What is the primary function of flagelli?
Movement
What is the primary function of the polysaccharide capsule?
Protection - prevents MAC or opsonisation molecules attacking
What types of bacteria release endotoxin?
Gram negative bacteria
What types of bacteria release exotoxin?
Gram positive and gram negative bacteria
What are endotoxins?
Component of bacteria’s outer membrane released when bacteria are damaged. Less specific and toxic to the host. Heat stable
What are exotoxins?
Proteins secreted that are specific and heat labile
What are plasmids?
Circular pieces of DNA that often carry genes for antibiotic resistance
Gram positive cocci can be divided into…?
Staphylococcus (clusters) and streptococcus (chains)
How do you distinguish between staphylococcus and streptococcus
Catalase test:
Staph = catalase +ve (gas bubbles)
Strep = catalase -ve (no change)
How do you further distinguish between staphylococci bacteria?
Coagulase test:
+ve = Staphylococcus aureus
-ve = all others (e.g. staph. epidermidis, staph. saprophyticus )
How do you further distinguish between streptococci bacteria?
Blood agar haemolysis:
Alpha = e.g. streptococcus pneumoniae
Beta = e.g. streptococcus pyogenes/agalactiae
Gamma = streptococcus bovis
How do you further distinguish between alpha haemolytic streptococci?
Optochin test:
Resistant = viridans strep
Sensitive = streptococcus pneumoniae
Give 4 examples of gram positive bacilli
- Bacillus cereus (B. cereus)
- Listeria
- Diphtheria
- Clostridium (C. diff)
Give 3 examples of gram negative cocci
- Neisseria gonorrhoeae (diplococci)
- Neisseria meningitidis (diplococci)
- Moraxella catarrhalis
How do you distinguish between gram negative bacilli?
MacConkey agar:
Pink (+ve) = lactose fermenting e.g. E.coli, klebsiella pneumoniae
White (-ve)= non lactose fermenting e.g. shigella, salmonella
How do you further distinguish between non lactose fermenting bacteria?
Oxidase test:
+ve = pseudomonas aeruginosa
-ve = shigella, salmonella, proteus
How do you differentiate between negative oxidase non-lactose fermenting bacteria?
- Cultivate on XLD agar
- Red with black centres = salmonella
- Only red = shigella
What is the oxidase test?
Test used to determine if a bacterium produces certain cytochrome c oxidases
What does it mean if gram negative bacilli have a H antigen?
They are motile and have a flagellum
What is a virus?
An infectious, obligate intracellular parasite comprising genetic material surrounded by a protein coat and/or membrane
When not inside an infected cell, viruses exist as … which consist of …
Virions which consists of genetic material (DNA or RNA) and a protein coat (capsid)
What shapes can viruses be?
Helical, icosahedral and complex
What is an enveloped virus?
Virus surrounded by a lipid coat derived from the plasma membrane of the host cell
Give 2 examples of enveloped viruses
- Influenza
- HIV
Give 2 examples of non enveloped viruses
- Adenovirus
- Parvovirus
What is a nucleocapsid?
The protein coat of a virus (capsid) and the nucleic acid of the virus
Are viruses living?
NO - they do not feed or respire and cannot reproduce independently
What are the 5 steps of virus replication?
- Attachment to specific receptor
- Cell entry
- Host cell interaction and replication
- Assembly of virion
- Release of new virus particles
Name 5 ways viruses can cause disease
- Direct destruction of host cells
- Modification of host cells
- ‘Over reactivity’ of immune system
- Damage through cell proliferation
- Evasion of host defences
Give an example of direct destruction of host cells
Poliovirus causes host cell lysis and death after a viral replication period of 4 hours
Give an example of modification of host cells
Rotavirus causes villi to atrophy and epithelial cells to flatten. This decreases the small intestine surface area and prevents nutrients (e.g. sugar) from being absorbed to cause a hyperosmotic state and profuse diarrhoea
Give an example of ‘over reactivity’ of immune system
Hepatitis B causes jaundice, pale stool, dark urine, RUQ pain, fever/malaise and itching
Give an example of damage through cell proliferation
Human papillomavirus (HPV) is associated with cervical, penile, anal, vaginal, vulval and head/neck cancer - the insertion and replication of its DNA leads to dysplasia and neoplasia which leads to cell proliferation and local/metastatic spread
Give 6 examples of evasion of host defences
- Latency
- Cell to cell spread
- Antigenic variability (ability to change surface antigens)
- Prevention of host cell apoptosis
- Down regulation of interferon and other intracellular host defence proteins
- Interference with host cell antigen processing pathways
What are protozoa?
Microscopic unicellular eukaryotes
What are the 5 major groups of protozoa?
- Flagellates
- Amoebae
- Sporozoa
- Cilliates
- Microsporidia
What is malaria transmitted by?
Female anopheles mosquitos
What are the 5 species of malaria?
- Plasmodium falciparum
- Plasmodium ovale
- Plasmodium vivax
- Plasmodium malariae
- Plasmodium knowlesi
How is malaria diagnosed?
Look for trophozoites on blood film under a light microscope
Describe the use of the thick film in diagnosing malaria
Works out if the patient has malaria or not
Describe the use of the thin film in diagnosing malaria
Works out what species of malaria the patient has
What are 4 signs of malaria?
- Anaemia
- Jaundice
- Hepatosplenomegaly
- Black water fever
What are 8 symptoms of malaria?
- FEVER
- Chills
- Headache
- Myalgia
- Fatigue
- Diarrhoea
- Vomiting
- Abdominal pain
Describe the lifecycle of malaria
- Mosquito bites infected person and ingests plasmodium gametocytes
- These develop into sporozoites
- Mosquito bites another person and injects the sporozoites
- The sporozoites infect hepatocytes
- Infected hepatocyte becomes a schizont
- The schizont bursts and infects RBCs (where plasmodium becomes trophozoites and rupture RBCs and reinfect others)
- Some trophozoites develop into gametocytes which are taken up by other mosquitos
Which is the most severe species of malaria?
Plasmodium falciparum
How are RBCs infected with plasmodium falciparum different?
Have proteinaceous knobs on the surface that bind to endothelial cells and other RBCs. This can cause small vessels to become obstructed causing hypoxia and microinfarcts
Name 4 ways you can treat malaria
- IV artesunate
- IV quinine and doxycycline
- Riamet
- PO chloroquine
Which species of malaria can lie dormant?
Plasmodium ovale and plasmodium vivax can form hypnozoites in the liver
What makes up the cell wall of fungi?
Chitin and glucans (polysaccharides)
How do fungi move?
By growing across or through structures OR by dispersion in air/water
What forms do fungi exist in?
- Yeast - single cell that divides via budding
- Moulds - form multicellular hyphae or spores
What are worms also known as?
Helminths
What are the 3 groups of worms?
- Nematodes (round worms)
- Trematodes (flat worms a.k.a flukes)
- Ceratodes (tapeworms)
What are nematodes and what are the types?
Round worms
- Intestinal variety (e.g. threadworm)
- Larva migrans
- Filarial tissue worms (sometimes considered to be a 4th group)
What are trematodes and what are the types?
Flat worms (flukes)
- Blood (cause schistosomiasis)
- Liver
- Lung
- Intestinal
What are ceratodes and what are the types?
Tapeworms
- Non invasive (e.g. sit in the bowel)
- Invasive (invade the body)
Can adult worms replicate inside the body?
Not without a period of development outside the body
What is the pre-patent period of worms?
Interval between infection and appearance of eggs/larvae in stool
What do glycopeptides (antibiotics) do?
Inhibit cell wall synthesis
How are intestinal nematodes transmitted?
From human to human via eggs or larvae (not usually infectious when first passed and has to undergo a period of development in the soil). Faecal-oral spread
How are worms usually diagnosed?
- Stool microscopy for eggs
- Seeing the worm itself (if big enough e.g. intestinal nematodes)
What do intestinal nematodes cause and how are they treated?
Malnutrition (travel around the body)
Treatment: Mebendazole, piperazine, pyrantel, levasimole
What is ground itch?
Papules and itching at the site of entry on the larvae (e.g. feet) caused by hookworm (intestinal nematode that travels around the body). The commonest cause of iron deficiency anaemia in the world
How is hookworm treated?
Mebendazole, pyrantel
What is enterobius vermicularis also known as?
Pinworm or threadworm (intestinal nematodes that don’t travel, goes from mouth straight through to anus but can poke into appendix or vaginal/uterus penetration)
What is hyperinfection syndrome?
Associated with auto-infection and an immunocompromised state e.g. due to advanced HIv or corticosteroid therapy
What are 3 symptoms of hyperinfection syndrome?
- Diarrhoea
- Weight loss
- Malabsorption
Give 2 examples of glycopeptides (antibiotics)
- Vancomycin
- Teicoplanin
What do beta lactams (antibiotics) do?
Inhibit cell wall synthesis by disrupting peptidoglycan synthesis
What are the 4 types of beta lactams (antibiotics)?
- Penicillins (e.g. amoxicillin, flucloxacillin, benzylpenicillin)
- Cephalosporins (e.g. cephalexin, cefotaxime)
- Carbapenems (e.g. estapenem, imipenem)
- Monobactams (e.g. aztreonam)
What do antibiotics e.g. trimethoprim and sulphonamides do?
Inhibit nucleic acid synthesis by inhibiting folate synthesis
What do antibiotics e.g. ciprofloxacin and fluroquinolones do?
Inhibit nucleic acid synthesis by inhibiting DNA gyrase
What do antibiotics e.g. rifamipcin do?
Inhibit nucleic acid synthesis by binding to RNA polymerase
What do antibiotics e.g. metronidazole do?
Inhibit nucleic acid synthesis by causing breaks in DNA strands
Which 4 types of antibiotics inhibit protein synthesis?
- Chloramphenicol
- Macrolides (e.g. clarithromycin, erythromycin)
- Tetracyclines (e.g. doxycycline)
- Aminoglycosides (e.g. gentamicin, streptomycin)
What do bactericidal antibiotics do?
Kill bacteria e.g. antibiotics that inhibit cell wall synthesis
What is a consequence of bactericidal antibiotics?
Can lead to the release of endotoxin (bits of cell wall) which cause an increase in antigenic load –> aggressive/dangerous inflammatory response
What do bacteriostatic antibiotics do?
Prevent growth of bacteria e.g. antibiotics that inhibit protein synthesis/DNA replication/metabolism
Why might bacteriostatic antibiotics be better?
Reduced toxin production and endotoxin surge less likely
Define minimum inhibitory concentration
Lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation
Give 4 examples of antibiotic resistance
- Change antibiotic target
- Destroy antibiotic
- Prevent antibiotic access
- Remove antibiotic from bacteria
Describe and give an example of changing antibiotic targets
- Bacteria change the molecular configuration of antibiotic binding sites or masks them
- E.g. flucloxacillin can no longer bind to PBP of staphylococci
Describe and give an example of destroying antibiotics
- Antibiotic destroyed or inactivated
- E.g. penicillins and cephalosporins have beta lactam rings which become hydrolysed by bacterial enzymes (beta lactamase) so that they cannot bind to PBP
What does PBP stand for?
Penicillin binding proteins (in bacteria)
Describe and give an example of preventing antibiotic access
- Bacterial membrane porin channel is modified e.g. in size, number, selectivity
- E.g. gram negative bacteria against glycosides
Describe and give an example of removing antibiotics from bacteria
- Proteins in bacterial membranes act as exports/efflux pumps to reduce the levels of antibiotics
- E.g. enterobacteriacae resistance to tetracyclines
Describe intrinsic resistance by bacteria
- Natural resistance
- All subpopulations of a species are equally resistant
Give an example of intrinsic resistance by bacteria
Gram negative bacteria have an outer membrane that cannot be penetrated by vancomycin
Describe acquired resistance by bacteria
- Bacteria which were previously susceptible obtain the ability to resist the activity of a particular antibiotic
- Only certain subpopulations of a species will be resistant
Give an example of acquired resistance by bacteria
- Spontaneous gene mutation
- Horizontal gene transfer (conjugation, transduction, transformation)
When diagnosing viral and bacterial infections, which swabs are best?
Viral - green swabs
Bacterial - charcoal swabs
How often do bacteria divide?
Every 30-60 minutes
What does the cell wall of mycobacteria contain?
Lipoarabinomannan
What may mycobacteria cause?
Meningitis
Antimicrobial resistance is spread by…
Plasmid mediated gene transfer
What does MRSA refer to and how is it usually treated?
Methicillin (flucloxacillin) resistant staphylococcus aureus - treated with glycopeptides (e.g. vancomycin)
What is the first line treatment for streptococcus pyogenes?
Penicillin
EBV infection can cause…
Infectious mononucleosis (mono)
What would tests of infectious mononucleosis show?
- Atypical lymphocytes on a blood film
- Detectable EBV IgM in serum
