Introduction to the rules of behavior and work in microbiological

laboratory
General rules of conduct in the laboratory
Respecting, the laboratory rules enables good results in routine and scientific research work,
but also avoids dangers such as:

1. The fire (burner)

2. Toxic fumes and toxic substances

3. Injuries caused by broken glass

Each student must wear a white coat, and put the excess clothes on the coat rack, not on the
table or chair.
It is not allowed to walk around the laboratory during classes or to sit at tables or inadequate
surfaces.
It is obligatory to report any irregularity, deficiency or damage as soon as possible.
Smoking is strictly forbidden in the laboratory.
Long hair must be tied at the back due to the risk of mechanical injury and flames.
Food and drink must not be brought into the laboratory.
Workbenches must always be clean without chemicals and devices that are not currently in use.
Broken glass, porcelain and other solid waste items should be stored in special baskets,
specially prepared for this purpose, separated from paper and soft waste.
Bottles with chemicals should always be kept clean because if their contents spill they could
become very slippery and easily fall out of your hands.
All chemicals should be clearly labeled even when in normal containers or cups.
It is obligatory to wash your hands before entering the laboratory as before leaving it!

INSTRUMENTS AND EQUIPMENT IN THE MICROBIOLOGICAL LABORATORY

OPTICAL LIGHT MICROSCOPE

INOCULATING LOOP
An inoculation loop, also called a smear loop, inoculation wand or microstreaker, is a simple tool used
mainly by microbiologists to pick up and transfer a small sample (inoculum) from a culture of
microorganisms, e.g. for streaking on a culture plate.

PETRI DISH
Shallow transparent lidded dish that biologists use to hold growth medium in which cells can be
cultured, originally, cells of bacteria, fungi and small mosses.

TUBES AND TEST TUBES

REFRIGERATOR THERMOSTAT AND AUTOCLAVE

VITEK SYSTEM

NEEDLES, SYRINGES, VACUTAINERS, MICRODILUTION PLATES

SWABS AND TUBES

SAMPLING FOR MICROBIOLOGICAL EXAMINATION
SPECIMEN SAMPLING
1. THE HUMAN BODY
Sterile regions:
1. central nervous system
2. cardiovascular system
3. parenchymal organs
4. bones and joints
5. muscle tissue
Regions colonized by normal microflora:
1. upper parts of respiratory tract
2. initial parts of urogenital tract
3. digestive tract
4. skin
2. WATER AND FOOD
3. AIR, LAND, WALLS, FLOORS, OBJECTS
4. STERILITY CONTROL IN LABORATORIES (INSTRUMENTS)

PRINCIPLES OF SPECIMEN SAMPLING FOR MICROBIOLOGICAL EXAMINATION

 Adequate sample
 From the right place (avoid contamination)
 At the right time
 Adequate amount
 Sterile equipment
 Before starting antibiotic therapy
 Give instructions to the patient
 Proper marking and transport

THROAT SWAB SAMPLING

NOSE SWAB SAMPLING

EYE SWAB SAMPLING

EAR SWAB SAMPLING

ENDOCERVICAL SWAB SAMPLING

URETHRAL SWAB SAMPLING

URINE SAMPLING- MICTURITION

A mid-stream urine sample means you don't collect the first or last part of urine that comes
out. This reduces the risk of the sample being contaminated with bacteria from: your hands. the
skin around the urethra, the tube that carries urine out of the body.
URINE CULTURE COLLECTION INSTRUCTIONS
 Wash hands thoroughly with soap and water, rinse and dry.
 Open the collection package but DO NOT TOUCH INSIDE OF CUP OR RIM. Open the
package of 3 towelettes. While seated on the toilet spread labia major (outer folds).
With the first towellete, wipe one side of the labia minora (inner fold) using a single
downward stroke. Discard towellete. With the second towellete repeat the procedure
on opposite side using a single downward stroke. Discard towellete. With the third
towelette, cleanse meatus (center area) with a single downward stroke. Discard
towellette.
 Remove lid carefully from the collection container, DO NOT TOUCH the inside of the
container or rim. Gently grasp the container.
 Begin to void urine, letting the first 20-25 ml pass into the toilet. Position the cup in the
stream of urine until the container is about one-half to two-thirds full. Finish voiding
into the toilet.
 After obtaining the urine specimen, screw the lid on tightly again being careful to avoid
touching inside the container or lid.
 Bring the specimen to the lab within 1 hour of collection or store refrigerated for up to
24 hours

URINE SAMPLING- CATHETERIZATION AND SUPRAPUBICAL PUNCTURE

PUS- SWAB, PUNCTURE, INCISION

SPUTUM SAMPLING

FECAL SAMPLING
BLOOD SAMPLING (BLOOD CULTURE, SEROLOGY)

PUNCTURE OF SYNOVIAL FLUID

CEREBROSPINAL FLUID SAMPLING

SAMPLING OF BONE MARROW

TISSUE BIOPSY
 IDENTIFICATION OF BACTERIA- MICROSCOPIC METHODS FOR STUDYING
MICROORGANISMS. WET MOUNT. GRAM STAINING
OBJECTIVES OF BACTERIOLOGICAL DIAGNOSTICS:
1. isolation and identification of infectious agent
2. susceptibility testing of agents to antimicrobial agents

IDENTIFICATION OF BACTERIA (DIRECT DIAGNOSIS)

 Morphological features
 Cultural characteristics
 Physiological-biochemical properties
 Antigenic properties
 Biological examination
 Gene testing

MORPHOLOGICAL FEATURES
Preliminary classification of bacteria into particular group
Microscopic properties:
1. Bacterial cell shape
2. Bacterial cell size
3. Bacterial cell arrangement
4. Bacterial staining characteristics
5. The presence of certain structural elements of the bacterial cell

BACTERIAL CELL SHAPE

 Ball-shaped, spherical (cocci)
 Rod-shaped (bacilli)
 Spiral
 Irregular
 Kidney shaped
 Candle flame

BACTERIAL CELL SIZE

 Genetically determined
 Depends on the conditions in which the bacterium is located (nutrient medium,
temperature, antibiotics)
BACTERIAL CELL ARRANGEMENT
Defined by the angle of the dividing planes and the conditions of the environment, as well as
the shape.
It indicates belonging to a certain genus.

BACTERIAL STAINING CHARACTERISTICS

Staining of bacteria depends on the chemical composition of their cells, but also on the
properties of the dyes themselves.
Staining provides better observation of the bacterial morphological characteristics.
STRUCTURAL ELEMENTS OF A BACTERIAL CELL
1. Capsule
2. Flagele
3. Fimbriae
4. Metachromatic beads
5. Spores

MICROSCOPY
1. Light microscope
 Wet mount
 Hanging drop
 Dark field microscopy
 Microscopy of colored
preparations
2. Fluorescent microscope
3. Phase contrast
microscope
4. Electron microscope

WET MOUNT
Observation of live, unstained
bacteria.
THE HANGING DROP METHOD
For longer observation of the preparation.

MICROSCOPY OF COLORED PREPARATIONS

1. DIRECT (from patient material)
 Sample quality
 Inflammation / colonization
 Type and percentage of bacterial cells
 Sometimes is enough for a diagnosis
2. FROM CULTURE
 Shape, size, layout, structure
 They are observed under conditions of homogeneous immersion

PHASE CONTRAST MICROSCOPE

Different cell structures are visible due to changes in the speed of light (different refractive
index).

PREPAIRING PREPARATIONS FOR STAINING- FIXATION

Fixation with flame or acetone / methyl alcohol.
STAINS AND STAINING OF BACTERIA
Colors:

1. Acid (eosin, acid fuchsin, safranin)

2. Alkaline (methylene blue)
3. Neutral
Staining:
1. Simple
2. Complex
 Differential (Gram)
 Special (Ziehl-Neelsen)

SIMPLE STAINING
1. Monochromatic (methylene blue)
2. Metachromatic (toluidine)
3. Negative contrast (nigrosine)

COMPLEX STAINING
1. Staining acc. to Gram – Gram staining
2. Staining acc. to Ziehl-Neelsen
3. Staining acc. to Hauser
4. Staining acc. to Neisser
5. Staining acc. to Olt

GRAM STAINING
Differential staining
Cell wall of Gram-positive bacteria Cell wall of Gram-negative bacteria
REQUIRED SUBSTANCES FOR GRAM STAINING
1. Crystal violet
2. Lugol's solution (Iodine)
3. Ethyl alcohol
4. Carbol fuchsin (safranin)
5. Water

Direct microscopic preparations (DMP)-Neisseriae Gonorrhoreae

DMP:Polymorphonuclear leukocytes and gram positive cocci

STAINED ACC. TO ZIEHL- NEELSEN
Differential acid fast staining for acid-alcohol resistant bacteria (Mycobacteriae)
Required substances:

 Carbol fuchsin
 Water
 3% HCl (hydrochloric acid) dissolved in 96% ethanol
 Methylene blue
 Water

STAINING ACC. TO HAUSER

Special staining – endospores
STAINING ACC. TO NEISSERU
Special staining – metachromatic granules Coyinebacterium diphtheriae

STAINING ACC. TO OLT

Special staining (safranin) - bacterial capsule
IDENTIFICATION OF BACTERIA II- BACTERIAL CULTURE MEDIA (PURPOSE, TYPES
AND CLASSIFICATION). CULTURAL AND BIOCHEMICAL IDENTIFICATION OF
BACTERIA
CULTURAL CHARACTERISTICS OF BACTERIA-NUTRIENT MEDIA
Conditions for culturing bacteria in vitro:
1. Water and electrolytes (Ca, Mg, K, Na, Fe, Mn)
2. Organic growth factors (carbohydrates, amino acids, purines, pyrimidines)
3. Oxygen (deep agar - aerobic, anaerobic)
4. Temperature (37 ° C)
5. pH (7-7,4) - Vibrio cholerae (8,2), lactobacilli (5,6)
6. Osmotic pressure (Enterococcus, Staphylococcus - media with 6.5% NaCl)

CLASSIFICATION ACCORDING TO OXYGEN REQUIREMENTS

OSMOTIC PRESSURE

BACTERIAL CULTURE MEDIA

Most bacteria can be cultivated on nutrient media.
This is not possible only with obligatory intracellular bacteria (chlamydia, rickettsiae).
Bacterial culture media are complexes of artificially united elements necessary for maintaining
the life of bacterial cells.
Objectives: isolation and identification of bacteria, their quantification and susceptibility testing
to antimicrobial drugs.

CLASSIFICATION OF BACTERIAL CULTURE MEDIA ON THE BASIS OF CONSISTENCY

1. Liquid (Broth) medium
2. Semisolid medium
3. Solid medium(agar)
4. Biphasic medium
BACTERIAL GROWTH ON LIQUID MEDIA
 Surface: ring, scrum, grains
 Blurring
 Sediment: granular, flaky, mucous
 Positive geotaxy phenomenon (streptococcus)

BACTERIAL GROWTH ON A SEMI-SOLID

MEDIUM (MOTILITY)

GROWTH OF BACTERIA ON A SOLID SUBSTRATE- BACTERIAL COLONIES

 The shape
 Size
 Edges
 Surface area
 Elevation
 Consistency
 The color
 Transparency
 Smell
 Hemolysis
CLASSIFICATION OF BACTERIAL CULTURE MEDIA ON THE BASIS OF PURPOSE/
FUNCTIONAL USE/ APPLICATION
1. Nonselective media
2. Selective media
3. Differential/ Indicator media
4. Enrichment media
5. Transport media

NONSELECTIVE MEDIA
1. Basal or General-Purpose Media - support the growth of most non-fastidious bacteria
 Peptone water, nutrient broth, and nutrient agar
2. Enriched media - Basal medium with addition of extra nutrients in the form of blood,
serum, egg yolk, etc,. used to grow nutritionally exacting (fastidious) bacteria
 Blood agar, chocolate agar, Loeffler’s serum slope
SELECTIVE MEDIA
Designed to suppress the growth of some microorganisms while allowing the growth of others.
With addition of antibiotics, dyes, chemicals, alteration of pH, or a combination of these.
Agar-based (solid) medium so that individual colonies may be isolated

 Thayer Martin Agar (to recover Neisseria gonorrhoeae contains antibiotics; vancomycin,
colistin, and nystatin)
 Mannitol Salt Agar and Salt Milk Agar (to recover S.aureus contains 10% NaCl)
 Potassium tellurite medium (to recover C.diphtheriae contains 0.04% potassium
tellurite)
 MacConkey’s Agar (for Enterobacteriaceae members contains bile salt that inhibits most
gram-positive bacteria)
 Lowenstein Jensen Medium (to recover M.tuberculosis is made selective by
incorporating malachite green)
 TCBS Agar (for isolating Vibrio cholerae from fecal specimens have elevated pH (8.5-8.6),
which inhibits most other bacteria)

DIFFERENTIAL/INDICATOR MEDIA
Different bacteria can be recognized on the basis of their colony color.
By incorporation of dyes or metabolic substrates, bacteria that utilize them appear as
differently colored colonies with the presence of indicator.

 Mannitol salts agar (mannitol fermentation = yellow)

 Blood agar (various kinds of hemolysis i.e. α, β and γ hemolysis)
 Mac Conkey agar (lactose fermenters, pink colonies whereas non- lactose fermenter
produces pale or colorless colonies)
 TCBS (Vibrio cholerae produces yellow colonies due to fermentation of sucrose)
ENRICHMENT MEDIUM
Liquid media used to increase the relative concentration of certain microorganisms in the
culture prior to plating on solid selective medium.
Serves to inhibit commensals in the clinical specimen

 Selenite F broth
 Alkaline peptone water

TRANSPORT MEDIA
Clinical specimens must be transported to the laboratory immediately after collection to
prevent overgrowth of contaminating organisms or commensals and maintain the viability of
the potential pathogens. This can be achieved by using transport media. Such media prevent
drying (desiccation) of a specimen, maintain the pathogen to commensal ratio, and inhibit the
overgrowth of unwanted bacteria.

CHROMOGENIC MEDIUM
PHYSIOLOGICAL AND BIOCHEMICAL CHARACTERISTICS OF BACTERIA
Because the clinical samples will most likely contain many microorganisms, both normal flora
and pathogens, it is important to isolate the pathogen in a pure culture using various types of
selective and differential media.

A Few Biochemical/Physiological Properties Used for identification of bacteria include: nutrient

utilization (carbohydrate utilization, amino acid degradation, lipid degradation), resistance to
inhibitory substances (high salt, antibiotics, etc.), enzyme production (catalase, coagulase,
hemolysins, etc.) and motility.

NUTRIENT UTILIZATION
1. Carbohydrate
 Monosaccharides (glucose, fructose, galactose, ribose)
 Disaccharides (sucrose, lactose)
 Polysaccharides (starch)
2. Polyhydric alcohols (glycerol, mannitol, sorbitol)
3. Amino acids (tryptophan, methionine) and proteins
AMINO ACID DEGRADATION TESTS
1. Indole production test (peptone water, indicator-Kovács reagent)
2. Hydrogen sulfide production test (indicator-FeC6H6O7, Pb (CH3COO) 2)
DEGRADATION OF CITRATE AND UREA
1. Simons citrate agar
2. Christensen's urea and Ferguson's urea

ANALYTICAL PROFILE INDEX- API

API is a classification of bacteria based on biochemical tests, allowing fast identification. This
system is developed for quick identification of clinically relevant bacteria.
Because of this, only known bacteria can be identified.

DEMONSTRATION OF THE PRESENCE OF BACTERIAL ENZYMES

1. The catalase test
2. The coagulase slide test - Cadness-Graves test (clumping factor)
3. Microtube coagulase test (4h)

THE CATALASE TEST

The catalase test tests for the presence of catalase, an enzyme that breaks down the harmful
substance hydrogen peroxide into water and oxygen. If an organism can produce catalase, it
will produce bubbles of oxygen when hydrogen peroxide is added to it.
THE COAGULASE SLIDE TEST
The coagulase slide test is used to identify the presence of bound coagulase or clumping factor,
which is attached to the cell walls of the bacteria. Bound coagulase reacts with the fibrinogen in
plasma, causing the fibrinogen to precipitate.

TUBE COAGULASE TEST

Tube coagulase test identifies whether an organism produces the coagulase, which causes the
fibrin of blood plasma to clot.

ANIMAL LAB TESTING

The only method of reproduction for bacteria that cannot be cultivated.
It is used for bacterial virulence testing, capsule production ability, detection of bacterial toxins.
ANTIGENIC PROPERTIES OF BACTERIA
 Fast identification method
 Classification of bacteria (group, species, type specific antigens)
 Immunological methods

GENE TESTING AND PCR

The technique is highly sensitive and produces results rapidly.
INTERPRETATION OF BACTERIOLOGICAL DIAGNOSTIC RESULTS
Waiting for the results is usually 2-3 days
When to start therapy?
Interpretation of results which derived from:

 Primarily sterile regions (false negative results - errors in taking, transporting or

processing the material / false positive results - contamination)
 Regions colonized by physiological flora (quantitative analysis, carrier state / clinical
picture / immune status, opportunistic infections / change of colonization site)

ANTIBIOGRAM
ANTIMICROBIAL DRUGS
Substances that kill microorganisms or prevent them from multiplying in a living organism are
called antibiotics and chemotherapeutics, or commonly called antimicrobial drugs.
Antibiotics can be divided into two classes based on their mechanism of action.
Bactericidal antibiotics kill bacteria; bacteriostatic antibiotics inhibit their growth or
reproduction.
They are used to treat or prevent bacterial diseases in humans
Antibiotics are natural, biological products of various fungi, while chemotherapeutics are
products of chemical synthesis.

IDEAL PROPERTIES OD ANTIMICROBIAL DRUG

 Selective toxicity
 Rapid achievement of therapeutic concentration at the site of action
 Keeping the drug in active form long enough
 Wide range of actions
 The structure of the drug is unsuitable for the development of resistance

MECHANISM OF ACTION OF ANTIBACTERIAL DRUGS

 Inhibition of cell wall synthesis
 Depolarization of the cell membrane
 Inhibition of protein synthesis
 Inhibition of nuclei acid synthesis
 Inhibition of metabolic pathways in bacteria (folate metabolism)
ANTIBIOTICS WHICH INHIBIT CELL WALL SYNTHESIS
1. Beta-lactams (target site - PBP)
 Penicillins
 Cephalosporins
 Carbapenems
 Monobacteria
2. Glycopeptides
 Vancomycin
3. Fosfomycin and cycloserine
ANTIBIOTICS WHICH DEPOLARIZE THE CELL MEMBRANE
1. Polymyxin and Colistin (disorganize the cytoplasmic membrane and lead to loss of
permeability)
2. Polyene antibiotics (bind to sterols)
 Nystatin
 Amphotericin B
3. Imidazoles (directly damage the cytoplasmic membrane)
 Miconazole
 Fluconazole
ANTIBIOTICS WHICH INHIBIT PROTEIN SYNTHESIS

Inhibitors of ribosomal subunits 30 S

 inhibit protein synthesis by binding to bacterial cell ribosomes and inhibiting translation

Inhibitors of ribosomal subunits 50 S

 inhibit protein synthesis by preventing peptidyl transferase from binding to the 50 S
subunit of the ribosome

Aminoglycosides (streptomycin)

Tetracyclines

Chloramphenicol

Macrolides (erythromycin)
ANTIBIOTICS WHICH INHIBIT NUCLEI ACID SYNTHESIS
DNA synthesis inhibitors (bind to DNA gyrase)
 Quinolones (nalidixic and pipemidic acid)
 Fluoroquinolones (ofloxacin, perfloxacin, norfloxacin, ciprofloxacin)

RNA synthesis inhibitors

 Rifampicin (binds to DNA-dependent RNA polymerase)

ANTIBIOTICS WHICH INHIBIT METABOLIC PATHWAYS IN BACTERIA (FOLATE

METABOLISM)
Sulfonamides (structural similarity to PABA)
Trimethoprim (inhibits dihydrofolate reductase and purine synthesis)
Combination of sulfamethoxazole and trimethoprim.

RESISTANCE
Congenital (lack of target structure)
Acquired
1. Nongenetic (M.Tuberculosis, L forms)
2. Genetic
 Chromosomal
 Extrachromosomal (plasmids, transposons)

MECHANISMS OF RESISTANCE
 Enzymatic destruction or inactivation of the drug
 Modification of the target enzyme
 Alteration of cell membrane permeability
 Alteration of ribosome structure
 Change in metabolic pathway
Bacterial resistance to drugs is a consequence of uncritical and irrational use of antibacterial
drugs for therapeutic and prophylactic purposes, but also the ability of bacteria to relatively
quickly develop mechanisms of resistance to each new drug.
ANTIBIOGRAM
Susceptibility testing of microorganisms to antibiotics and chemotherapeutics.
First antibiotic tests:

 Direct instillation of antibiotics on the substrate

 Application of antibiotic solution in agar wells

BASIC STANDARDIZED ANTIBIOGRAM PROCEDURES

1. Diffusion method
2. Dilution methods
 Agar dilution
 Broth dilution
3. Combined methods
 E test
 Regression line

CATEGORIES OF BACTERIAL SUSCEPTIBILITY TO ANTIBIOTICS

Qualitative (S-I-R)
1. Sensitive
2. Intermediate sensitive
3. Resistant

Quantitative (mg / μl)

1. MIC - The Minimum Inhibitory Concentration is defined as the lowest concentration of
an antimicrobial agent that is bacteriostatic (prevents the visible growth of bacteria)
2. MBC - The Minimum Bactericidal Concentration is the lowest concentration of an
antibacterial agent required to kill a bacterium over a fixed, somewhat extended period,
such as 18 hours or 24 hours, under a specific set of conditions.

DIFFUSION METHOD OF ANTIBIOGRAM

It is based on the ability of antibiotics to diffuse through a solid nutrient medium.
Concentration at a certain distance depends on:
1. Quantities of the substance
2. Diffusion coefficient
3. Distances from the initial diffusion site
PROCEDURE FOR PERFORMING THE DIFFUSION METHOD
The method consists of placing paper disks saturated with antimicrobial agents on a lawn of
bacteria (purificated culture - 10cfu/ml) seeded on the surface of an agar medium (Müeller
Hinton agar), incubating at 35-37 °C the plate overnight (18-24h), and measuring the presence
or absence of a zone of growth inhibition around the disks.

DISC DIFFUSION METHOD

ADVANTAGES AND DISADVANTAGES OF THE DIFFUSION METHOD

Advantages:
 Simplicity and speed the procedure
 Simultaneous examination of the susceptibility of one bacterial strain to a number of
antibiotics

Disadvantage:
 Impossibility of direct reading of MIC

DILUTION METHOD OF ANTIBIOGRAM

 Using a series of double dilutions of antibiotics made in liquid or solid nutrient media
 Broth dilution method
 Agar dilution method
 Incubate at 35-37 ° C, 18-24h
 Reading MIC and MBC values
BROTH DILUTION METHOD PROCEDURE
The broth/tube dilution test is the standard method for determining levels of microbial
resistance to an antimicrobial agent.
Serial dilutions of the test agent are made in a liquid microbial growth medium which is
inoculated with a standardized number of organisms and incubated for a prescribed time. At
the end of the incubation period (generally 18-24 hours), the tubes are visually examined for
turbidity and the MIC value is read.
To determine the MBC, the dilution representing the MIC and at least two of the more
concentrated test product dilutions are plated and enumerated to determine viable CFU/ml.
The MBC is the lowest concentration that demonstrates a pre-determined reduction (such as
99.9%) in CFU/ml when compared to the MIC dilution.
ADVANTAGES AND DISADVANTAGES OF BROTH DILUTION METHOD
Advantages:
 Possibility of determining MICs and MBCs that help in choosing the appropriate
antibiotic and dosage

Disadvantages:
 The complexity of the procedure
 Susceptibility of one bacterial strain to one antibiotic
 The final result after 48 hours

AGAR DILUTION
The agar dilution method involves the incorporation of
varying desired concentrations of the antimicrobial agent into
an agar medium (molten agar medium), habitually using serial
two-fold dilutions, followed by the inoculation of a defined
microbial inoculum onto the agar plate surface.
The plates containing the dilutions of antimicrobial agents are
spot inoculated with a loop calibrated to deliver 1–2 μl.
The lowest concentration of antimicrobial agent at which complete inhibition of bacteria occurs
is the MIC.
ADVANTAGES AND DISADVANTAGES OF AGAR DILUTION METHOD
Advantages:
 Possibility to determine the value of MIC
 Simultaneous susceptibility testing of several bacterial strains (15-50)

Disadvantages:
 The complexity of the procedure
 Impossibility of routine determination of MBK values

COMBINATION OF DIFFUSION AND DILUTION METHOD OF ANTIBIOGRAM

1. E test - epsilometer test
2. Regression line

E TEST METHOD
A plastic strip with a predefined gradient of one antibiotic is applied onto an inoculated agar
plate. After 18-24 hours incubation, a drop-shaped inhibition zone intersects the graded test
strip at the inhibitory concentration of the antibiotic.
The intersection of the lower part of the ellipse-shaped growth inhibition area with the test
strip indicates the MIC value.
PROPER CHOICE OF ANTIBIOTICS
 Knowledge of bacterial susceptibility
 Include antibiotics with different mechanisms of action in the antibiogram
 Take into account the pharmacodynamic properties of antibiotics
 Adjust the choice of antibiotics to the type of patient material
 Take into account the clinical signs, age and immune status of the patient
 Tolerant bacterial strains - MBK / MIK> 32

IMMUNODIAGNOSTIC METHODS IN MICROBIOLOGICAL DIAGNOSTICS-

SEROLOGICAL REACTIONS
SEROLOGICAL REACTIONS
Antigen-antibody reactions which can be observed in vitro.

ANTIGENS
Bacteria and their components and products
(enzymes, toxins)

Normal and abnormal components of serum,

plasma and other body fluids (immunoglobulins,
albumins, ceruloplasmin, components of the
complement system, C reactive protein, alpha-
fetoprotein).
SEROLOGICAL REACTIONS
Serological reactions can be applied in two ways:

1. in the diagnosis of disease by testing the serum (antibodies) of the patient against
known antigens and
2. to identify an organism (or other antigen) by testing it against known antisera
(antibodies)
Based on the created complex between antigen and antibody, an unknown component (Ag or
Ab) can be detected.

APPLICATIONS OF SEROLOGICAL REACTIONS

In microbiology, serologic tests are used to determine if a person has antibodies against a
specific pathogen (bacteria, virus, parasite or fungus), or to detect antigens associated with a
pathogen in a person's sample.
In transfusion medicine for blood typing and typin of histocompatible leukocyte antigens.
In immunology to diagnose autoimmune disorders by identifying abnormal antibodies directed
against a person's own tissues or abnormal serum components

TYPES OF SEROLOGICAL REACTIONS

1. Agglutination reactions
2. Precipitation reactions
3. Reactions involving labeled antigens or antibodies
4. Complement-fixation reactions

IMMUNOAGGLUTINATION REACTION
Immunoagglutination (often simply called
agglutination) is a laboratory diagnostic test
based on the reaction between a corpuscular
antigen and the matching specific antibody,
wherein the antigen is insoluble, or represent
an integral part of a large insoluble particle.
In this reaction, a large number of antibody and
antigen molecules cross-link (agglomerate) in a
big branched immune complexes and, as a
result, aggregate called agglutinate is formed that can be detected by the naked eye (or
optionally with the magnifying glass).
All antibody isotypes can agglutinate antigens, but IgM antibodies have the most prominent
capacity for agglutination, since they are pentamers and have 10 binding sites for antigen

In addition to the class of antibody, the effectiveness of agglutination is influenced by the

amount of antigen and antibodies in the reaction.
Optimal agglutinate formation occurs when the amounts of antigen and antibody are
approximately equal (so called zone of equivalence).

If either the antigen or the antibody is in excess, they will react, but very small complexes will
form that do not clump to form visible agglutination resulting in false negative result.

The lack of agglutination at high concentrations of antibodies is traditionally called prozone

effect (or prozone phenomenon).
This problem can be solved by diluting the tested sample, which reduces the amount of
antibodies, so that the equivalence between the antigen and the antibody can be achieved.

1. Primary stage – specific reaction between antibodies and antigenic determinants on

the particle surface (weak binding)
2. Secondary stage – agglutination (firm binding)
 Electrolyte concentration
 pH
 Temperature
 Time
 Particle size and their electrostatic charge
 Polyvalent antibodies
APLICATION OF IMMUNOAGGLUTINATION
 Determination of specific antigens
 Determination of titer of specific antibodies in serum
 The titer represents the reciprocal value of the highest dilution of the sample resulting
in a positive reaction (visible agglutination)

IMMUNOAGGLUT INATION METHODS

1. Direct - the antigen (antigenic determinant) is an integral part of a large particle (e.g.
bacterial surface molecule) that is directly agglutinated by antibodies
2. Indirect (passive) - the antigen is a soluble molecule passively adsorbed or chemically
attached to the surface of a large insoluble particle (erythrocytes, polystyrene inert
particles or latex microbeads) that becomes passive carrier of this antigen

DIRECT AGGLUTINATION
1. Gruber test

2. Widal test

3. Wile-Felix test

4. Wright test

5. Detection of microorganisms or antibody titers

INDIRECT AGGLUTINATION
 Antipenicilin antibodies
 Anti DNA autoantibodies
 Rheumatoid factor

INDIRECT (PASSIVE) AGGLUTINATION AND REVERSE PASSIVE AGGLUTINATION

Reverse passive agglutination - a type of agglutination reaction in which known antibody is
bound to a carrier particle instead of the antigen.
COOMBS TEST
A Coombs test (or antiglobulin test) is a special form of immunoagglutination used for the
detection of anti-erythrocyte antibodies that bind to red blood cells but do not lead to their
agglutination. Such antibodies occur in a variety of diseases such as hemolytic anemia
(autoimmune or caused by medication) or hemolytic disease of the newborn. The latter disease
can be caused by transplacental passage of maternal anti-RhD IgG antibodies from Rh negative
mother's bloodstream that is sensitized to the Rh positive fetus.
1. Direct - examination of the child's erythrocytes (Rh +)
2. Indirect - female antibody testing (Rh-)

IMMUNOPRECIPITATION
Immunoprecipitation (also called
precipitin reaction or precipitin test) is
a laboratory diagnostic test based on
the precipitation (deposition) of the
soluble antigen (macromolecule) by
the matching specific antibody (often
called precipitin) due to the formation
of large insoluble immune complexes
The result of the reaction is a
precipitate, which can be easily
detected (usually with the bare eye)
and its formation only occurs when
optimal concentration ratio of antigen and antibody (approximately equal amounts) is achieved
(the zone of equivalence).
IMMUNOPRECIPITATION METHODS
1. Methods in liquid medium
 Interfacial ring test
 Nephelometry
2. Methods in semisolid medium – gel
 Double gel immunodiffusion
 (Single) radial immunodiffusion
 Immunoelectrophoresis
 One-dimensional double electroimmunodiffusion (countercurrent electrophoresis)
 One-dimensional single electroimmunodiffusion (rocket electrophoresis)
3. Quantitative, semiquantitative and qualitative tests

IMMUNOPRECIPITATION
Both types of methods are based on the diffusion of the soluble antigens and/or antibodies in
appropriate medium, whereby the diffusion in the gel occurs more slowly.
The precipitate in the liquid media is formed relatively quickly (often within 10 minutes), while
the precipitation in the gel takes much longer time (sometimes up to 72 hours). Therefore, the
latter method is sometimes performed by using an electric current, with the aim to accelerate
the diffusion and formation of precipitates (electroimmunodiffusion method).

METHODS IN LIQUID MEDIUM

1. Interfacial ring test
2. Nephelometry

INTERFACIAL RING TEST

Ascoli reaction - detection of B. anthracis antigen
NEPHELOMETRY
Nephelometry is an analytical chemistry technique used to
measure the amount of turbidity or cloudiness in a solution
caused by the presence of suspended insoluble particles.
When directed through a turbid solution containing
suspended solid particles, light is transmitted, absorbed
(blocked) and scattered (reflected off the particles). The
amount of scattered light depends on the size, shape and
concentration of the insoluble particles in solution, as well as
on the incident wavelength of light.

METHODS IN SEMISOLID MEDIUM-GEL

Gel – made of agar, agarose or polyacrylamide
Characterized by formation of clearly visible precipitation line at the site where concentration
ratio of antigen and antibody reach equivalence and it is stable for longer period of time

Simple diffusion methods

 Double gel immunodiffusion
 (Single) radial immunodiffusion
 Immunoelectrophoresis

Methods by using electric current

 One-dimensional double electroimmunodiffusion (countercurrent electrophoresis)
 One-dimensional single electroimmunodiffusion (rocket electrophoresis)
DOUBLE GEL IMMUNODIFFUSION
Double gel immunodiffusion is often used for detection of an antigen orspecific antibody in a
tested sample. This assay is based on the migration of both soluble antigen and antibody
(hence the name “double“).
In this technique, the formation of the precipitation line in the zone of equivalence indicates
the presence of tested antigen (or antibody) in the sample and the result is considered positive.
If the sample does not contain tested antigen/antibody, no complex formation will occur and
the absence of precipitin
line is reported as
negative.
1. Qualitative and
semiquantitative
method
2. Elek's test
(diphtheria toxin)
ELEK'S
Elek's test is an In vitro immunoprecipitation (immunodiffusion) test to determine whether or
not a strain of Corynebacterium diphtheriae. A test strip of filter paper containing diphtheria
antitoxin is placed in the center of the agar plate. Strains to be tested (patient’s isolate), known
positive and negative toxigenic strains are also streaked on the agar’s surface in a line across
the plate and at a right angle to the antitoxin paper strip.
Antitoxin diffuses away from the strip of filter paper where as toxin produced by toxin-
producing strains diffuse away from growth. At the zone of equivalence a precipitin line is
formed.
RADIAL IMMUNODIFFUSION
The radial immunodiffusion (RID test) is
quantitative method, which is based on a
diffusion of the soluble antigen through a
gel that contains incorporated uniformly
distributed antibody (given that only the
antigen diffuses, this technique is
sometimes called single radial
immunodiffusion assay).
In the zone of equivalence an insoluble
precipitate in the shape of a circle is
formed (antigen is placed in a well that is
punched out of the gel medium and
diffuses in all directions).
When such a circular precipitation line is
formed, the diameter of the circle and the
amount of applied antigen are in direct
proportion, which can be used for determining the unknown concentration of antigen in the
sample, by comparison with the standards.
IMMUNOELECTROPHORESIS
Immunoelectrophoresis is defined as the separation and identification of proteins based on
differences in electrical charge and reactivity with antibodies.
When an electric current is applied to a slide layered with gel, the antigen mixture placed in
wells is separated into individual antigen components according to their charge and size.
Following electrophoresis, the separated antigens are reacted with specific antisera placed in
troughs parallel to the electrophoretic migration and diffusion is allowed to occur. Antiserum
present in the trough moves toward the antigen components resulting in the formation of
separate precipitation arcs in 18-24 hrs.

SERUM ELECTROPHORESIS
 All major proteins except immunoglobulins components migrate toward the anode
 Immunoglobulins migrate towards cathode
ONE-DIMENSIONAL DOUBLE ELECTROIMMUNODIFFUSION (COUNTERCURRENT
ELECTROPHORESIS)

 Detection of cryptococcal, meningococcal and hemophilus antigens

 Much faster and 10 times more sensitive than double immunodiffusion
ONE-DIMENSIONAL DOUBLE ELECTROIMMUNODIFFUSION (ROCKET
ELECTROPHORESIS)
Rocket immunoelectrophoresis is one-dimensional quantitative immunoelectrophoresis.
This fast and sensitive method has been used for quantitation of human serum proteins before
automated methods became available.

REACTIONS INVOLVING LABELED ANTIGENS OR ANTIBODIES

These reactions enable the detection of antigen or antibody using a reagent to which an
appropriate label is attached (radioactive isotope or enzyme).
Radioisotope - 125I
Enzymes - peroxidase, phosphatase, avidin-biotin
 1. Soluble phase immunoassays
 Radioimmunoassay (RIA)
2. Solid phase immunoassays
 Enzymoimmunoassay (ELISA)
3. Immunohistochemical tests
 Direct immunofluorescence
 Indirect immunofluorescence
 Flow cytofluorography

RADIOIMMUNOASSAY (RIA)
Detection of radiolabeled antigen
ENZYMOIMMUNOASSAY (ELISA)
In ELISA test antigen or antibody are in solid phase. Various antigen-antibody combinations are
used, always including an enzyme-labeled antigen or antibody, and enzyme activity is measured
colorimetrically. The enzyme activity is measured using a substrate that changes color when
modified by the enzyme.
Faster than RIA.
Example:

 Enzyme - alkaline phosphatase

 Substrate - p-nitrophenylphosphate
 Product - p-nitrophenol (yellow color)

DIRECT ELISA
A target protein (or a target antibody) is immobilized on the surface of microplate wells and
incubated with an enzyme-labeled antibody to the target protein (or a specific antigen to the
target antibody). After washing, the activity of the microplate well-bound enzyme is measured.

INDIRECT ELISA
A target protein is immobilized on the surface of microplate wells and incubated with an
antibody to the target protein (the primary antibody), followed by a secondary antibody against
the primary antibody. After washing, the activity of the microplate well-bound enzyme is
measured.
Although indirect ELISA requires more steps than direct ELISA, labeled secondary antibodies are
commercially available, eliminating the need to label the primary antibody.
SANDWICH ELISA
An antibody to a target protein is immobilized on the surface of microplate wells and incubated
first with the target protein and then with another target protein-specific antibody, which is
labeled with an enzyme. After washing, the activity of the microplate well-bound enzyme is
measured. The immobilized antibody (orange) and the enzyme-labeled antibody (green) must
recognize different epitopes of the target protein.

ENZYMOIMMUNOASSAY (ELISA)
 Indirect - detection of Rubeol antibodies
 Sandwich - HBsAg detection
IMMUNOFLUORESCENCE
Immunofluorescence (IF) is a common laboratory technique, which is based on the use of
specific antibodies which have been chemically conjugated to fluorescent dyes (fluorochromes-
fluorescein and rhodaine) or fluorophore
1. Primary (direct) immunofluorescence
2. Secondary (indirect) immunofluorescence

PRIMARY (DIRECT) IMMUNOFLUORESCENCE

Primary, or direct, immunofluorescence uses a single antibody that is chemically linked to a
fluorophore. The antibody recognizes the target molecule and binds to it, and the fluorophore
it carries can be detected via microscopy.
This technique has several advantages over the secondary (or indirect) protocol below because
of the direct conjugation of the antibody to the fluorophore. This reduces the number of steps
in the staining procedure making the process faster and can reduce background signal by
avoiding some issues with antibody cross-reactivity or non-specificity.
However, since the number of fluorescent molecules that can be bound to the primary
antibody is limited, direct immunofluorescence is less sensitive than indirect
immunofluorescence.

SECONDARY (INDIRECT) IMMUNOFLUORESCENCE

Secondary, or indirect, immunofluorescence uses two antibodies; the unlabeled first (primary)
antibody specifically binds the target molecule, and the secondary antibody, which carries the
fluorophore, recognises the primary antibody and binds to it.
Multiple secondary antibodies can bind a single primary antibody. This provides signal
amplification by increasing the number of fluorophore molecules per antigen.
This protocol is more complex and time consuming than the primary (or direct) protocol above,
but it allows more flexibility because a variety of different secondary antibodies and detection
techniques can be used for a given primary antibody.
FLUORESCENCE ACTIVATED CELL SORTING
Fluorescence activated cell sorting (FACS) of live cells separates a population of cells into sub-
populations based on fluorescent labeling. Sorting involves more complex mechanisms in the
flow cytometer than a non-sorting analysis. Cells stained using fluorophore-conjugated
antibodies can be separated from one another depending on which fluorophore they have been
stained with.
COMPLEMENT-FIXATION REACTIONS
In complement-fixation reactions a sample of serum is exposed to a particular antigen and
complement in order to determine whether or not antibodies to that particular antigen are
present. The nature of complement is to react in combination with antigen–antibody
complexes.
1. Complement-fixation test
2. Cytotoxic test
3. Complement titration test
COMPLEMENT FIXATION TEST
The CF test involves 2 basic principles :

1. Complement (C) is irreversibly bound (fixed) by certain classes of antibody-antigen

complexes (certain classes of antibodies do not fix the complement). The degree of
fixation is governed by the relative concentration of antibody or antigen
2. The lysis of sheep red blood cells that have been sensibilized with hemolysin
(antibodies) is dependent upon the presence of unbound complement
The CF test is interpreted as follows :

Antibody present=no hemolysis

Antibody absent=hemolysis

CYTOTOXIC TEST
COMPLEMENT TITRATION TEST
Quantification of total hemolytic activity of the complement system.

IMMUNOCHROMATOGRAPHY
Immunochromatographic tests rely principally on the capture of the target antigen (or
sometimes antibodies) from various specimens. The assay utilizes antibodies mounted on a
paper strip or a nitrocellulose membrane as the immobile capture antibody (test area).

MOLECULAR METHODS IN MICROBIOLOGICAL DIAGNOSTICS

1. Western blot

2. Southern blot

3. Dot blot

4. In situ hybridization

5. Northern blot

6. PCR

WESTERN BLOT
Western blot is the analytical technique used in molecular biology and immunogenetics to
detect specific proteins in a sample of tissue homogenate or extract.
Western blot technique uses three elements to achieve its task of separating a specific protein
from a complex:
1. separation by size,
2. transfer of protein to a solid support,
3. and marking target protein using a primary and secondary antibody to visualize
SOUTHERN BLOT
A Southern blot is a laboratory method used to detect specific DNA molecules from among a
many other DNA molecules.
Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter
membrane and subsequent fragment detection by probe hybridization.
DOT BLOT
Dot and slot blotting are simple techniques for immobilizing bulk unfractionated DNA on a
nitrocellulose or nylon membrane. Hybridization analysis can then be carried out to determine
the relative abundance of target sequences in the blotted DNA preparations.

IN SITU HYBRIDIZATION
In situ hybridization is a laboratory technique in which a single-stranded DNA or RNA sequence
called a probe is allowed to form complementary base pairs with DNA or RNA present in a
tissue or chromosome sample. The probe has a chemical or radioactive label attached to it so
that its binding can be observed.

NOTHERN BLOT
A northern blot is a laboratory method used to detect specific RNA molecules among a mixture
of RNA.
PCR (POLYMERASE CHAIN REACTION)
(PCR), a technique used to make numerous copies of a specific segment of DNA quickly and
accurately.
The polymerase chain reaction enables investigators to obtain the large quantities of DNA that
are required for various experiments and procedures in molecular biology, forensic analysis,
evolutionary biology, and medical diagnostics.
PCR is a three-step process that is carried out in repeated cycles.
The initial step is the denaturation, or separation, of the two strands of the DNA molecule. This
is accomplished by heating the starting material to temperatures of about 95 °C. Each strand is
a template on which a new strand is built.
In the second step the temperature is reduced to about 55 °C so that the primers can anneal to
the template.
In the third step the temperature is raised to about 72 °C, and the DNA polymerase begins
adding nucleotides onto the ends of the annealed primers. At the end of the cycle, which lasts
about five minutes, the temperature is raised and the process begins again.
The number of copies doubles after each cycle. Usually 25 to 30 cycles produce a sufficient
amount of DNA.