Microbiology Practicals Flashcards
How is a gram stain prepared?
Come In And Stain:
Heat fix bacteria onto the slide
Crystal violet - primary stain
Iodine
Acetate/Alcohol - decolourise
Safranin - Counterstain
What colour do gram positive and gram negative bacteria stain?
Gram positive - Purple
Gram Negative - Pink
What is blood agar?
agar with horse/sheeps blood
used for growing lots of bacteria
least selective
What is chocolate agar?
heated to 80C for breakdown of haem & release of nutrients/factors that grow organisms w/ fastidious growth requirements [H.influenzae]
What is CLED?
differentiates between organisms in urine & allows classification of lactose fermenting (yellow) & non lactose fermenting (blue) gram-negative bacilli
What is MacConkey Agar?
grows gram negative bacilli (it has bile salts that inhibit the growth of gram positive). Has lactose & red dye
lactose fermenting = pink colonies
non lactose fermenting = white colonies
What is Gonococcus Agar?
Contains growth factors to promote the growth of Neisseria species as well as antibiotics & antifungal agents to inhibit the growth of other organisms.
What is Sabouraud’s Agar?
used to culture fungi. Has antibiotics to inhibit bacterial growth.
What is XLD Agar?
used to isolate Salmonella and Shigella [non lactose fermenting, gram negative]
Salmonella - Red with black centre (produces H2S)
Shigella - Red only
What colour will Staphylococcus aureus appear on blood agar and what colour would other staphylococcus species appear?
S.aureus - gold
S.other - white (S.epidermidis/ S.saprophyticus)
What are the sterile sites of the body?
Blood
CSF
pleural fluid
Peritoneum
Joints
Urinary Tracts
Lower Respiratory Tract
How can you tell the difference between various Gram Positive bacteria?
+tve Catalase Test = Staphylococcus:
(if positive then do Coagulase Test:)
+tve = S.aureus
-tve = S.epidermidis/S.saprophyticus
-tve Catalase Test = Streptococcus
(if negative then do Haemolysis on blood agar)
alpha haemolysis (partial/green) = requirement for Optochin Test
Optochin Sensitive = S.pneumoniae
Optochin Resistant = S. viridans group
Beta haemolysis (full) = requirement for Lancefield grouping via serology)
A = S.pyogenes
B = S.agalactiae
A,C, G = Tonsilitis, pharyngitis, skin infection
B = Neonatal sepsis + meningitis
Gamma Haeomolgysis (none) = Enterococcus
How can you tell the difference between various Gram Negative bacteria?
MacConkey Agar for lactose fermentation Test:
+tve lactose fermentation = E.coli/Klebsiella
-tve Lactose fermentation = Requirement to do Oxidase Test
Oxidase Test:
+tve Oxidase = Pseudomonas aeruginosa
-tve Oxidase = Requirement to do XLD test:
Colonies on XLD which are Red with black centers = Salmonella
Colonies on XLD which are Red only = Shigella
What are the 2 main approaches to diagnosing viral infection?
Electron Microscopy / PCR
Serology Testing - ELISA, IF, Complement Fixation Test.
What is an advantage and disadvantage to EM testing?
Very specific and sensitive but too expensive and too long
What is an advantage and disadvantage to PCR?
Very fast but high risk of false positives as it is very sensitive and so can easily be contaminated.
What is serology?
Detection of antibody responses in the serum - Especially IgG and IgM
IgM implies acute/new infection
IgG implies later infection
What virus causes shingles and how does it present?
Varicella Zoster Virus (VZV)
Red painful rash conferred to a single dermatome
What virus causes Glandular fever (infectious mononucleosis)?
Epstein Barr Virus (EBV)
Ix: FBC - Atypical lymphocytes
Serology for EBV Igs - ELISA Test
What is a differential Diagnosis to Glandular fever and how is this ruled out?
S.pyogenes throat infection - Both EBV and S.pyogenes present with purulent (pus) infection over the tonsils
Take black charcoal swab to rule out S.pyogenes
What other Sx can be caused by EBV?
Tonsilitis,
Cough
Fever
Splenomegaly
What is BALF?
Bronchoalveolar lavage fluid
Fluid collected from the lungs
Bronchi are washed and this fluid is sent for analysis using PCR
What condition can cytomegalovirus cause?
CMV colitis
Characteristic finding is OWL EYE INCLUSION BODIES
What is the treatment for CMV infection?
IV ganciclovir
What HIV markers can be found in the blood
HIV Igs
HIV RNA
p24 antigen
How may a patient present with meningitis?
Headache
Fever
Photophobia
Neck stiffness
What Ix are required for suspected meningitis?
Take CSF sample.
CSF –> Increased WCC + protein
Determine type of meningitis based on CSF glucose, CSF Protein, type of WCC and CSF appearance.
If viral then use PCR to determine specific type
What antibody would you expect to find in an acute EBV infection?
IgM antibody
What antibody would you expect to find upon subsequent exposure to EBV or its reactivation?
IgG
VCA IgG
EBNA IgG
What are the uses of qPCR?
Quantitative PCR:
Look for the presence or absence of DNA/RNA
To quantify the level of virus in a tissue
What tests are performed to process a CSF sample?
Cell count and differential
Gram stain for organisms
qPCR for virus and bacterial pathogens
Protein and Glucose
What are the HIV markers tested for in the blood?
HIV Igs
HIV RNA
p24 antigen
What are the next steps if the HIV test yields:
+ lab ELISA
- Lab ELISA
+tve Lab ELISA:
Doctor explains +tve result to patient.
Needs a second confirmation blood sample
-tve Lab ELISA:
Confirmatory test with a different assay
What is the most common cause of meningitis in neonates?
Group B Strep - Strep Agalactiae
E.coli
Listeria
What is the most common cause of meningitis in infants?
S. pneumoniae
N. Meningitidis
H. influenza - now rare due to vaccine
What is the most common cause of meningitis in adults?
S. pneumoniae
N meningitidis
What is the most common cause of meningitis in the elderly?
S. pneumoniae
N. meningitidis
Listeria monocytogenes
What is the most common cause of Encephalitis?
Viral - HSV-1
(accounts for 95% of cases)
How are infections of the CNS diagnosed?
CSF analysis:
Cell count
Gram stain
Protein
Glucose
Culture on BA/Chocolate Agar and PCR
Nose/throat swab - Charcoal and Green viral swabs
What are the results of CSF analysis for bacteria?
Cells
CSF appearance
Protein
Glucose
Cells: Neutrophilia
CSF appearance: Cloudy yellow
Protein: High
Glucose: Low (<50%)
What are the results of CSF analysis for Viruses?
Cells
CSF appearance
Protein
Glucose
Cells: Lymphocytosis
CSF appearance: Clear
Protein: Normal - mildly elevated
Glucose; normal
What are the results of CSF analysis for Fungi?
Cells
CSF appearance
Protein
Glucose
Cells: Lymphocytosis
CSF appearance: Yellow and fibrinous
Protein: High
Glucose: Low (<50%)
What is the major risk factor of Meningococcal Septiciaemia?
Septic DIC
How does Meningococcal septicaemia present?
Non-Blanching Purpuric Rash W/ Meningism signs
What is the Treatment for meningococcal Septicaemia?
Immediate IM Benzylpenicillin
Then Transfer to Hospital
What is the Treatment for Listeria suspected meningitis?
IV Amoxicillin
What must be done after a diagnosis of Meningitis?
Inform PHE
Close Contact Tracing - Single dose Ciprofloxacin Prophylaxis
What are the investigations and Mx for Encephalitis?
Typically Temporal Lobe affected on CT
CSF analysis (LP) and PCR to confirm HSV-1
Tx - IV Acyclovir
What are the key samples to collect in Pneumonia, TB or COPD infection?
Bloods and Sputum
What is the common bacteria involved in pneumonia?
S. pneumoniae - Causes Typical Pneumonia
Cultured on Blood Agar - Alpha Haemolytic and optochin Sensitive
What is Typical Pneumonia?
Invades alveolar cavity and causes a productive rust coloured purulent sputum with a fever and headache
What is the Treatment for Typical Pneumonia?
Amoxicillin - Covers most Strep and S. aureus sensitive strains
What is the most common cause of Atypical Pneumonia?
Legionella pneumophilia - Suspected in Severe CAP and Recent Travel (spain)
How are atypical pneumonias treated?
Macrolides - Erythromycin/clarithromycin
What is the first line treatment for Legionella pneumonia?
1st Line - Clarithromycin
Inform PHE too
What are some other causes of Atypical Pneumonia?
chlamydia psittaci
Mycoplasma Pneumonia
Chlamydia pneumonia
Coxiella
What are the main bacteria involved in COPD exacerbations?
Haemophilus Influenzae (gram neg bacillus)
Tx with Amoxicillin (if beta lactamase negative) or Co-amoxiclav (if Beta lactamase positive)
S. pneumonia:
Tx with Amoxicillin
How can TB be detected?
Stain: Ziehl Neelsen - AFB detected
(not gram stain due to high mycolic acid content of TB prevents gram stain uptake)
Auramine phenol fluorescence and Microscopy
Solid TB Culture - Lowenstein Jensen Agar
(TB Replicates every 15-20hrs so may take 6 weeks to grow
NAAT/PCR - Much faster than solid culture
Histologically - Granuloma w. Central Necrosis (caseating)
What cause of pneumonia is common in HIV Px?
Pneumocystis Pneumonia caused by Pneumocystis Jirovecii
Shows cysts of Jirovecci Fungus
Tx with Co-Trimoxazole + Prednisolone if Type I Respiratory Failure present
What Organisms often colonise the respiratory tract in bronchiectasis patients?
P. aeruginosa
Tx with Tazocin - Tazobactam + piperacillin
S. pneumonia
H. Influenza
Tx with Amoxicillin
What is the common skin infection of the lower limb?
Cellulitis
DDx - DVT
What are the cardinal Signs of Inflammation?
Calor
Dolor
Rubor
Tumor
Loss of function
What is the pathogenesis of Cellulitis?
Breach in skin (Eczema, Athletes foot, Diabetic Foot)
Pathogen enters the soft tissues through the breach
Causes infection and inflammation
What are the common Cellulitis organisms?
Group A Strep - S. pyogenes
S. aureus
What is the Presentation of Impetigo?
Crusty lesions on chin and Cheek of a young person
(acute infection in children)
Which is itchy and grows as they Scratch it
What is the Presentation of Impetigo?
Crusty lesions on chin and Cheek of a young person
(acute infection in children)
Which is itchy and grows as they scratchWhat
What is the Presentation of Impetigo?
Crusty lesions on chin and Cheek of a young person
(acute infection in children)
Which is itchy and grows as they scratchWhat
What swab is used to diagnose impetigo?
Black Charcoal Swab
What is the treatment for Impetigo?
S. pyogenes - Benzylpenicillin
S. aureus - flucloxacillin
MRSA - Vancomycin or Teicoplanin
What is the treatment for Cellulitis?
Give Empirical Abx until pathogen ID w/ culture then Review.
Empirical Abx: Flucloxacillin
What are the 3 key features of taking a blood culture for Cellulitis?
Take good volumes of blood
Take from more than one site
Take on more than one occasion
What are the features of a lower UTI?
LUTs:
Storage - FUNI
Voiding - SHITS
What are the features of an upper UTI?
Pyelonephritis
Loin pain
Fever
Nausea and Vomiting
What are the main UTI Organisms?
KEEPS
Klebsiella
E.coli (UPEC 80% of cases)
Enterobacter
Proteus Mirabilis
S. Saprophyticus /S. epidermidis
What are the main causes of UTIs?
Obstruction
BPH
Renal Stones
Catheters
Sexual intercourse
Renal Tumours
What is the first line investigation for a suspected UTI?
Urine Dipstick:
+ leukocytes
+ nitrites
+/- Protein
What level of bacteria is significant for a UTI on Culture?
10^5
What is the treatment for an uncomplicated UTI?
Nitrofurantoin (50mg 4x Daily) for 3 days
What type of sample should urinalysis never be done on?
Catheterised sample urine:
Bacteria is likely present regardless of infection or not compared to Mid-Stream which should be reliable as the urinary tract is normally sterile
What is the treatment for E. coli Pyelonephritis?
Co-amoxiclav for 7 days or Nitrofurantoin for 14 days
Why should pregnant women have their urine screened?
Screen for Pre-Eclampsia
High BP and Proteinuria in pregnancy when otherwise well
What is the most common cause of Infective Endocarditis?
S. aureus
What is the treatment for Campylobacter infection?
Ciprofloxacin / Clarithromycin
What is the treatment for C. difficile infection?
Stop C Abx
Vancomycin 125mg 4x Daily for 10 days
