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  • The best microbiologist in the world will not be able to accurately identify an infectious agent from a poorly acquired or transported specimen, and it is the patient who will suffer

Roles of the clinician and the microbiologist in the diagnosis of infection

  • Role of the clinician
    • Collection of specimen
    • Storage and transport of specimen
    • (The clinician should understand microbiology and know the tests to order)
  • Role of the microbiologist
    • Processing of specimens
    • Identification of the infectious causative agent
    • Provision of antibiotic susceptibilities (if relevant)

Considerations for a clinician when collecting a specimen

  • Whenever possible, specimens must be collected prior to antibiotic therapy
    • Once the patient is on antibiotics, microbes may have reduced in number, making it harder to identify the causative organisms
    • Exceptions: life-threatening conditions e.g. meningitis, septicaemia (microbes multiplying in blood stream => septic shock (TNFa, IL-1) => organs shut down => die)
  • The specimen must be representative of the condition (i.e. taken from the right place)
    • Saliva or a throat swab is unlikely to lead to the isolation of an organism causing pneumonia
    • We don’t want to know what is in the mouth, but what is in the lung – they need to cough up phlegm
  • Aseptic technique
  • Use of sterile swabs or containers
  • Correct transport conditions for specimens
  • Labelling specimens
  • Completing the request form

Aseptic technique

Is making sure you do not contaminate the specimen

  • When a needle or canula is inserted through the skin to collect a specimen, the patient’s skin must be swabbed with an antibacterial agent prior to collection
  • The NO touch technique
  1. Use clean gloves etc
  2. Swab the area
  3. Once you have swabbed the area make sure you do NOT touch:
    • The swabbed area
    • The needle/canula (If you touch the needle/canula before putting it into the patient, you could infect them)
    • The top of the bottle into which the specimen will be inoculated
  4. Collect the sample
  5. Don’t touch the lid of the blood culture bottle

Blood culture

  • Blood for culture must be collected in a syringe and then inoculated into blood culture bottles (BCBs)
  • BCB contain enrichment broth to increase the numbers of any bacteria that may be present in the blood
    • Blood is normally sterile
    • If there is an infection, then there are relatively small numbers of organisms in the blood
    • Since there aren’t many organisms in the blood, we can’t grow it directly onto a plate, so we enhance the growth with BCB
    • Incubate at 37 C until the alarm goes off in the incubator (tests turbidity). This indicates that you have enough microbes present
    • Then subculture: grow the specimen on an agar plate to identify microbes and test for antibiotic susceptibility

Collection of urine

  • We need to collect a mid stream urine (MSU) sample
    • First part and last part of the stream aren’t wanted
    • In many urinary-tract infections, it’s not whether an organism is present, it is how many are present. This is because many urinary tract infections are caused by abnormal proliferation of normal flora deep in the urinary tract
    • The initial stream washes out normal flora present in the urethra, and we get a good reading after that
    • (However, in testing for STDs, we sometimes need the early stream urine)

Transport and storage of the specimen

  • Specimens collected using a swab
    • e.g. wound with pus or penile/vaginal discharge etc
  • Potential problems:
    • Desiccation causes death of pathogen
    • Toxicity causes death of pathogen
    • Overgrowth of normal flora may prevent isolation of the pathogen (by producing toxic metabolites that kill the sample)

Solution to this problem is:

Transport media

  • Transport media maintain the pathogen in a viable state
  • Particularly important for fastidious organisms (that need particular nutrients)
  • Specimens are collected using a swab
    • e.g. wound with pus or penile/vaginal discharge etc
  • Then put the swab into a cylindrical container, at the bottom of which is the medium. The end of the swab will fall into the medium
  • Transport media contains
    • a semi-solid agar that prevents specimen drying out
    • charcoal that inactivates toxic material
  • Examples of transport media:
    • Amie’s
    • Stuart’s
    • Amie’s charcoal
  • In transport media, organisms can last for 2-3 days (which is much longer than without the medium)

Transport temperature

  • For most specimens it is essential that they are stored and transported under particular conditions
  • Swabs: 4C or room temperature, depending upon the suspect pathogen
  • Urine: Must be stored and transported at 4C
    • At 4C most microbes will live but not multiply
    • Stops normal flora and pathogen from multiplying
    • The number of organisms per litre is a critical factor in diagnosis (if normal flora proliferate and get too far up the urinary tract then you get an infection)
  • Cerebrospinal fluid (CSF): Store at room temperature (do NOT refrigerate)
    • Organisms causing meningitis die at 4C
  • Hence the choice of storage temperature is organism-dependent

Labelling of the specimen container

  • Each specimen must have a label showing:
    • The date and time of collection
    • Name, sex and D.O.B. of the patient
    • The type of specimen
  • If these are not present, the specimen will be discarded

The request form

This must provide full details:

  • Name and details (address etc)
  • Clinical details
    • Very important so they can choose which organisms to look for based on clinical history
  • Antibiotic therapy
  • Collection date and your (the doctor’s) name
  • Specimen description – swab/pus/drain/aspirate/fluid/tissue/CSF/faeces
  • Investigation required
    • Tell them what to look for!

The steps from specimen collection to microbiological diagnosis

  1. Patient consultation
  2. Specimens
    • If urine, faeces, blood, CSF, go to 3
    • If blood (serum), go to 7
  3. Microbiology laboratory – detection of microbes or their products
  4. Identification of pathogen
  5. Antibiotic susceptibility testing
  6. Final report
  7. Serology laboratory – detection of antibodies
  8. Report

  • Note that Microbiology and Serology are sometimes done in the same laboratory
  • The final report is sent out to you (the doctor)
  • Sometimes you can get preliminary results based on early tests

Processes at the microbiology laboratory

  1. Specimen arrives
  2. Register specimen and patient’s details
  3. Macroscopic observations
  4. Microscopic observations
  5. Rapid test for antigens [Sometimes 3-5 can be used to provide a preliminary report]
  6. Cultures set up
  7. Identification of causative agent
  8. Antibiotic sensitivity testing if relevant
  9. Serology if relevant

[6-9 take longer to be carried out and come in a final report]

Macroscopic observations

  • Urine
    • Cloudy or presence of red blood cells, indicates infection
  • CSF
    • Turbid – likely to be bacterial (but can never assume)
    • Clear – more likely to be viral
    • Bloody – often because of a “bloody tap” – spinal tap collection that has resulted in some blood being taken
  • Sputum
    • Mucoid? Colour

Microscopic observations

Microscopy may in some cases allow preliminary identification of pathogen

  • Whether direct microscopy is useful or not depends on whether the specimen is collected from a sterile site (normal flora may interfere)
  • If the specimen is taken from a sterile site, then YES: direct microscopy is useful
  • Sterile sites include:
    • Blood
    • CSF
    • Bladder
    • Serous fluids
    • Tissues, bladder
    • Lower respiratory tract
    • Stomach (except for helicobacter pylori)
  • If the specimen is from a site with normal flora, then the usefulness depends on whether the causative agent can be discriminated from normal flora (in most cases it is difficult)
    • In some cases, the pathogen has a unique morphology (gram stain and shape) and so can be identified
  • Sites with a normal flora include:
    • Upper respiratory tract
    • Female genital tract
    • Urethra
    • Intestinal tract
  • Note: there are no gram negative cocci native to the vagina
    • Presence of PMNs also indicates infection
    • Yeast will form pseudohyphae (because the yeast is in good conditions)

Microscopic identification of microorganisms

  • Bright field microscopy (light microscopy)
    • Gram stain is used for bacteria. Allows determination of morphology
    • Wet preparation is used for urine
      • Haemacytometer (counting station)
      • Lots of PMNs and a high amount of bacteria imply a bacterial infection
      • Lots of epithelial cells => not MSU.
  • Electron microscopy
    • Viruses (though it’s not really done, unless it’s a new virus)
    • May allow determination of viral morphology

The Gram Stain

  • The gram stain also allows us to determine the shape of the bacterium
  • See elsewhere for what the gram stain is and why some are negative and some are positive

Rapid tests for antigen, toxin or gene sequence detection

  • Antigen detection involves detection of bacterial soluble carbohydrate antigens
    • Agglutination with antibody coated latex particles or RBC
    • Antigen-antibody reaction results in agglutination
    • We introduce an antibody that has a latex bead attached
    • Latex bead lets you see the agglutination
    • Eg
      • Strep pneumoniae
      • Haemophilus influenzae
      • Neisseria meningitides
      • Cryptococcus neoformans
  • Toxin detection
    • E.g. Clostridium difficile – Antibiotic associated diarrhoea
  • Polymerase chain reaction (PCR)

PCR Assays

  • Polymerase chain reaction
    • The polymerase chain reaction (PCR) is a scientific technique inmolecular biology to amplify a single or a few copies of a piece of DNAacross several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
    • Typically, PCR consists of a series of 20-40 repeated temperature changes, called cycles, with each cycle commonly consisting of 2-3 discrete temperature steps, usually three (Fig. 2). The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.[8]
    • Initialization step: This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR.[9]
    • Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.

o Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.

    • Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activitytemperature at 75–80 °C,[10][11] and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.
    • Final elongation: This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
    • Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.
    • To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder(a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products (see Fig. 3).

PCR assays are available for more rapid diagnosis of some pathogens, however they cannot determine antibiotic susceptibility.

  • Bacterial examples:
    • Chlamydia trachomatis
    • C. pneumoniae
    • Mycobacterium tuberculosis
    • Mycoplasma pneumoniae
    • Neisseria gonorrhoeae
  • Viral examples
    • Herpes simplex virus (HSV)
    • Human papilloma virus (HPV)
    • Cytomegalovirus
  • PCR assays that monitor viral load
    • E.g. HIV, Hepatitis C, Hepatitis B

(NB: Gel elecrophoresis:

  • Agarose PCR gel used to visualise gene sequences
  • Apply an electric current, then stain it
  • Molecular weight “ladder” is set up)

Culture of bacteria and fungi

  • Clinical notes are essential
  • Based on these notes, we decide which media we might use – “what to check for”
  • Prior to culture, the likely pathogens must be determined as this determines:
  • Choice of media
  • Temperature of incubation
  • Atmospheric conditions
  • Length of time for culture

Culture media

Basal medium

  • Basic nutrients microbes need to grow
  • Nutrient broth
  • Beef extract
  • Yeast extract
  • Peptone
  • NaCl
  • Add agar (agar-agar from algae, 1.5%)
  • Clear colour

Enriched media

  • Sheep blood agar (SBA) – add blood to the agar
  • Chocolate blood agar (CBA) – heat blood slightly to release NAD and haem factors for growth
  • Example:
  • Haemophilus influenzae is fastidious … needs particular factors
    • SBA – no growth
    • CBA - growth

Selective media

  • Used to “choose” particular types of organisms
  • E.g. Sabouraud’s agar (to “choose” yeasts or fungi)
  • High concentration of glucose, low pH
  • Bacteria have limited or no growth
  • Yeasts and fungi have good growth (they can live at low pH)
  • E.g. Media containing antibiotics cuts back normal flora (use it if you suspect yeast and fungi)

Differential media (alpha, beta, gamma)

  • A differential medium distinguishes between different types of bacteria based on some characteristic of the bacteria that is growing on it. Typically there is a colour change that results from certain bacterial metabolic products reacting with substances or chemicals that have been added to the media.
  • Blood agar
  • Allows differentiation of bacteria based on haemolysis
  • Alpha – green colour – partially lysed blood cells
  • Beta – see right through it – completely lyse blood cells (looks yellow – not sure if it is supposed to)
  • Gamma – no lysis – looks normal

MacConkey Agar (MAC)

  • MacConkeys Agar is a special bacterial growth medium that is selective for Gram- bacteria and can differentiate those bacteria that are able to ferment lactose.
  • Nutrient agar
  • Bile salts – selective
  • Lactose – differential
  • pH indicator
  • Example:
  • Lactose fermenter (LF) (e.g. E. Coli) – red colour (showing a lactose fermenter)
  • Non-lactose fermenter (NLF) (e.g. Salmonella sp.) – cream colour
  • This medium is both selective and differential


Correct technique for streaking a plate

To be able to observe the pathogen and then identify it, we need to streak for single colonies

  1. Use bacterial loop
  2. Flame it
  3. Cool it
  4. Dip it in inoculant
  5. Streak it

Single colonies are required for tests etc. Each single colony originated from a single organism (hence the same species).

Incubation conditions

Incubation temperature

  • Optimum temperature to incubate most pathogens is 37C (although some are different)

pH requirements

  • pH 6.5-7

Optimum pH for most bacteria

  • pH 5-6

Moulds and yeasts

  • pH <4
  • Limited number of bacteria e.g. Lactobacilli

Gaseous requirements for bacterial growth

  • Aerobic – require oxygen for growth
  • Anaerobic – cannot grow in the presence of oxygen
  • Facultative aerobe/anaerobe – can grow in the presence or absence of oxygen
  • Microaerophilic – Can only grow in reduced oxygen (<20% atmospheric O2)
Anaerobic culture apparatus

Growing anaerobes

  • Grow them in an anaerobic culture apparatus
  • Envelope contains sodium bicarbonate and sodium borohydride. Add water – causes breakdown (producing CO2 and H2)
  • Lid has O-ring gasket
  • Top has a clamp with a clamp screw, below which are palladium catalyst pellets
  • Palladium catalyst uses H2 to convert O2 to water
  • Produces an anaerobic environment
  • Anaerobic indicator (methylene blue)
  • Following incubation, a range of bacteria may be present if the specimen comes from a site with a normal flora
  • # Colonies = # of species of bacteria
  • Subculture of putative (suspected) pathogen leads to pure culture

Identification of bacterium

Once we have a pure culture of the pathogen we can identify the bacterium

  • Gram reaction – gram positive (purple/black) or gram negative (pink)
  • Cell morphology – rods or cocci
  • Cultural characteristics (differential/selective medium)
  • Biochemical characteristics
  • Antigen differences (latex beads etc)

Biochemical identification tests

  • Specific enzymes (does it have specific enzymes?)
  • Catalase (e.g. different between streptococci and staphylococci)
  • Oxidase
  • Urease (e.g. will show up for helicobacter pylori)
  • Don’t mix cultures – otherwise we may get mixed results (e.g. see slides)

Antibody detection

  • Commonly used for the diagnosis of viral infections (not electron microscopy)
  • Look for antibodies to suspect virus in patient’s serum
  1. Gather serum
  2. Use ELISA (e.g. IgG test)
  • Must collect acute and convalescent phase sera and look for a 4X rise in titre
  • Acute IgG titre: 4
  • Convalescent IgG titre: 32
  • If there is only one serum sample, then what antibody would be indicative of active infection? IgM (first one that forms – right away)

Diagnosing a throat infection

  • Specimen collection
  • Specimen taken by touching a sterile swab to the back of the patient’s throat
  • Tongue depressor may be used
  • Must not touch other areas of the mouth
  • Transport media is required if there is any delay in getting to the lab (Stuart’s transport media)
  • Ensure that the specimen is labelled and the request form is completed
  • In the microbiology lab:
  • Direct gram stain
  • Culture of specimen
  • Media used – blood agar
  • Incubation – both aerobic and anaerobic
  • Kept at 37C for 24 hours
  • Following incubation, look for:
    • Beta haemolytic colonies
    • Gram stain beta haemolytic colony
    • Gram positive cocci
    • Catalas negative
    • Sensitive to bacitracin
  • Identification: Streptococcus pyogenes