Diagnostic Microbiology: Specimen Analysis

• Diagnostic microbiology involves studying specimens from patients suspected of infections.
• The study aims to assist clinicians in definitive diagnoses and decisions regarding antimicrobial therapy.
• Clinicians should understand specimen collection techniques and the principles behind laboratory analysis.
• Diagnosing infectious diseases involves decisions and actions by many people.

Diagnostic Cycle

• The diagnostic cycle starts with a clinical request and the provision of clinical information.
• Collection and transport of appropriate specimens comes next.
• Laboratory analysis is then performed.
• Finally, interpretation of the microbiology report and its use occur.

Clinical Requests

• Steps in the diagnostic cycle include clinical request, provision of clinical information, collection/transport of specimens, lab analysis, and interpretation of the microbiology report and the use of information.
• The first stage in the diagnostic cycle comprises the specimen and its request form.
• Factors influencing the quality of the specimen should be noted.
• It is futile to sample the patient for pathogens if they are not suffering from a microbial infection.

Provision of Clinical Information

• Specimens should be collected before antibiotic therapy, if possible.
• The patient should be seriously ill.
• The patient should beimmunologically compromised.
• The patient should not be responding to a specific antibiotic
• Microbiologists select appropriate tests based on clinical information.
• Such information includes age, clinical condition, illness onset date, current/recent antibiotic therapy, allergies, and previous specimen history, which are important for investigations.

Collection and Transport of Specimens

• Collect appropriate specimens, as some organisms (anaerobes, viruses) do not survive long at room temperature.
• Organisms like coliforms and staphylococci may multiply at room temperature, leading to misleading analysis results.
• Transport specimens in appropriate media, otherwise dehydration or exposure to aerobic conditions can cause the specimen to be unusable.
• Transport media has to be compatible with its organisms.
• Virus specimens should be transported in viral transport medium.
• Safe, robust containers avoid contamination.

Laboratory Analysis

• Specimen is obtained from dental abscess
• Specimens are received and analysed in diagnostic microbiology laboratories.
• A smear of the specimen is made, followed by Gram staining.
• Specimen is then inoculated on 2 blood agar plates for culture under aerobic and anaerobic conditions.
• Plates are inspected for growth.
• Shapes and colony types are noted for subculture.
• Infections can be polymicrobial or monomicrobial organism origin.
• Putative pathogens are isolated by subculturing onto fresh blood agar plates.
• Those plates are then incubated at 37°C for 24-48 hours.
• A pure culture of the pathogen is harvested and identified using biochemical reactions, selective media, or specific antibody reactions.
• Antibiotic sensitivity tests can be performed on the mixed growth.
• The microbiologist can issue a provisional report after 2 days, but the final report may take longer (5 days).

Interpretation of Results

• While the interpretation reports may be straightforward, in situations, the clinician may need contact the microbiologist for antibiotic guidance or sampling.

Laboratory Methods

• Laboratory methods are categorized into non-cultural or cultural.
• Non-cultural methods include microscopic methods and detection of microbes by probing for their genes using molecular tools.
• Cultural methods use solid or liquid media for bacterial and fungal growth.
• Cultured cells from animals and humans are used for viral growth.
• Immunological methods identify organisms (Quelung reaction) and detect antibodies in patients' body fluids.

Microscopic Methods

• Microscopic methods include stained and wet films
• Bright-field or standard microscopy routinely used in diagnostic microbiology.
• Lesions, stained smears are tested under oil immersion objective (x100) using a x10 eyepiece, multiplying the object to x1000.
• Wet films are examined with a dry objective (x40) for motility of bacteria.
• Dark-ground microscopy illuminates the specimen obliquely using a special condenser.
• Organisms appear bright against a dark background.
• Fluorescence microscopy uses emission principles involving the use of ultraviolet light onto bacteria or Auramine stained cells to help detect microbial antigens.
• Electron microscopy uses light waves replaced by a beam of electrons. This allows for virions to be seen, and used in diagnostic virology.

Gram Stains

• Bacteria are stained by results of gram staining
• Gram straining requires a smear to be fixed with crystal violet and washed
• Bacteria are then decolorized with gram iodine, and the samples that test positively are washed immediately.
• Lastly carbolfuchsin is added for 30s, which is washed with water, and blot dried

Ziehl-Neelsen Technique

• Ziehl-Neelsen is used to stain bacteria in samples like tubercle bacilli
• They are difficult to strain by gram method and possess thick wall layers
• The Ziehl-Neelsen is applied via Carbolfuchsin at 5 min, washed
• Decolorize it the aid of acid and alcohol, then counterstain with methylene blue.

Molecular Tools

• These are the detection of microbes by probing for their genes
• Very small bacterial numbers can be detected with the standard polymerase chair reaciton

Solid & Liquid Media

• Bacteria grow well on artificial media
• Blood agar, a nonselective medium. However, when chemicals are added to media to suppress growth, these become selective media.
• The addition of bile salts helps isolate enterobacteria from a stool sample.
• Thiosulphate citrate bile sucrose (TCBS) agar allows the yellow fermenter vibrio cholera, or green V. para hemolyticus to grow
• The main constituents of bacteriological media are water, agar (from seaweed), growth-enriching constituents (yeast extract, meat extract), and blood.

Solid Media

• The preparation of solid media, all ingredients are added
• Molten agar is dispensed when warm in either petri dishes or test tubes
• The molten agar is plated solid agar in bottles.

Liquid Media

• Promote growth or small numbers contaminates by antibiotics, which is then diluted.
• Aseptic broth is commonly used media for Liquid.

Transport Media

• Specimens are transported clinic with a transport medium, to the laboratory. Such helps to maintain viability in Bacteriological media, such as Stuart transport medium

Atmospheric Requirements and Incubation

• Following inoculation, the agar plates need incubated
• Aerobically: by either addition of 10% CO2 to increase the growth of human pathogens
• Anaerobically: with strict anaerobes to only grow in hydrogen
• Anaerobic can be produced, with environmental oxygen replaced by Nitrogen
• The body is incbated at 37 Degrees and fungi grow at ambient temperature

Bacterial and Viral Identification

• Is inspection of the colonial characteristics, size, shape, elevation and the type of hemolysis.
• Next is examination of microscopy and stain
• The process concludes at growth conditions

Biochemical Tests

• Each bacterial species has a distinctive biochemical profile valuable for identification
• Test inlcude sugar, enzymnes, with reactions like the Staphyloccus auerus

Commercial Method & ELISA

• These identify with each strain of bacterial with a Analytical Profile Index
• It can be done commercially
• ELISA also detects a spectrum where small samples are taken of each bacterial.
• ELISA can be performed in numerous steps

Determination of Antimicrobial Agents

• Laboratories test to determine antimicrobial therapy
• Antimicrobial Agents are tested by In clinical and microbiology

Susceptibility Results

• Results are obtained with the therapy
• It can be qualitatively and quantitativly to determine appropriate
• This assessment is known Mic and BCD Theses in Viro
• Involves inhibition which gives a growth
• Laboratory results is a a process where a drug's effect can be measured in 24-48 hours
• This process includes Kirby bauer Method and Mueller hin agar

Isolate Identification

• Isolate is one of several ways to determine the origin
• Includes Direct microscopy. a speedy test technique that test if there is any virus.
• Isolation Is for viruses that do not grow or support single culturing
• Lastly Virus specific antibodies can have virus antigens

Tissues

• Tissues display rounding, forming that can be tested via. electron microscopy
• Tests includes examination via stook or serology
• The serology tests include.immunofluorescence
• Tissue Culture cells are cheap in both cultures. such as with MKC- 9 or baby kidney cells.