Title: Diagnosis of Infectious Diseases
1Diagnosis of Infectious Diseases
2Laboratory Investigation of Microbial infections
- Examining specimens to detect isolate and
identify pathogens - 1- Microscopy
- 2- Culture techniques
- 3- Biochemical reactions
-
- 4- Serological identification
-
- 5- Molecular biology techniques
-
- 6- Bacteriophage typing
31- Microscopy
- Microorganisms can be examined microscopically
for - a- Bacterial motility
- Hanging drop method
- A drop of bacterial suspension is placed
between a cover slip and glass slid - b- Morphology and staining reactions of bacteria
- Simple stain methylene blue stain
-
- Gram stain differentiation between Gmve and
Gmve bacteria - . Primary stain (Crystal
violet) - . Mordant (Grams Iodine mixture)
- . Decolorization (ethyl
alcohol) - . Secondary stain (
Saffranin) - Ziehl-Neelsen stain staining acid fast
bacilli - . Apply strong carbol
fuchsin with heat - . Decolorization (H2SO4 20
and ethyl alcohol
- . Counter stain (methylen
blue)
42- Culture Techniques
- Culture media are used for
- - Isolation and identification of pathogenic
organisms - - Antimicrobial sensitivity tests
- Types of culture media
- a- Liquid media
- - Nutrient broth meat extract and
peptone - - Peptone water for preparation sugar
media - - Growth of bacteria detected by
turbidity - b- Solid media
- - Colonial appearance
- - Hemolytic activity
- - Pigment production
5Types of solid media
- 1- simple media
- Nutrient agar
- 2- Enriched media media of high nutritive
value - . Blood agar
- . Chocolate agar
- . Lofflers serum
- 3- Selective media allow needed bacteria to
grow - . LowensteinJensen medium
- . MacConkeys agar
- . Mannitol Salt Agar
- 4- Indicator media to different. between lact.
and non lact. ferment - . MacConkeys medium
- . Eosine Methlyne blue Agar
- 5- Anaerobic media for anaerobic cultivation
- . Deep agar, Robertsons Cooked Meat
Medium
6Colonial appearance on culture media
- Colony morphology
- . Shape . Size . Edge of
colony . Color - Growth pattern in broth
- . Uniform turbidity
- . Sediment or surface pellicle
- Pigment production
- . Endopigment production (Staph.
aureus) - . Exopigment production (Ps.
aeruginosa) - Haemolysis on blood agar
- . Complete haemolysis (Strept.
Pyogenes) - . Partial haemolysis
(Strept. Viridans) - Growth on MacConkeys medium
- . Rose pink colonies (Lactose
fermenters) - . Pale yellow colonies (Non
lactose fermenters)
73- Biochemical Reaction
- Use of substrates and sugars to identify
pathogens - a- Sugar fermentation
- Organisms ferment sugar with production of
acid only - Organisms ferment sugar with production of
acid and gas - Organisms do not ferment sugar
- b- Production of indole
- Depends on production of indole from amino
acid tryptophan - Indole is detected by addition of Kovacs
reagent - Appearance of red ring on the surface
- e- H2S production
- Depends on production H2S from protein or
polypeptides - Detection by using a strip of filter paper
containing lead acetate -
83- Biochemical Reaction (cont.)
- c- Methyl red reaction (MR)
- Fermentation of glucose with production
of huge amount of acid - Lowering pH is detected by methyl red
indicator - d- Voges proskaurs reaction (VP)
- Production of acetyl methyl carbinol from
glucose fermentation - Acetyl methyl carbinol is detected by
addition KOH - Color of medium turns pink (positive)
-
- e- Action on milk
- Fermentation of lactose with acid
production - Red color if litmus indicator is added
93- Biochemical Reaction (cont.)
- f- Oxidase test
- Some bacteria produce Oxidase enzyme
- Detection by adding few drops of colorless
oxidase reagent - Colonies turn deep purple in color
(positive) - g- Catalase test
- Some bacteria produce catalase enzyme
- Addition of H2O2 lead to production of gas
bubbles (O2 production) - h- Coagulase test
- Some bacteria produce coagulase enzyme
- Coagulase enzyme converts fibrinogen to
fibrin (plasma clot) - Detected by slide or test tube method
- i- Urease test
- Some bacteria produce urease enzyme
- Urease enzyme hydrolyze urea with
production of NH3 - Alklinity of media and change color of
indicator from yellow to pink -
104- Animal pathogenicity
- Animal pathogenicity test
- Animals commonly used are guinea pigs,
rabbits, mice - Importance of pathogenicity test
- - Differentiate pathogenic and non pathogenic
- - Isolation organism in pure form
- - To test ability of toxin production
- - Evaluation of vaccines and antibiotics
11Serological identification
- A- Direct serological tests
- - Identification of unknown organism
- - Detection of microbial antigens by using
specific - known antibodies
- - Serogrouping and serotyping of isolated
organism - B- Indirect serological tests
- - Detection of specific and non specific
antibodies - (IgM IgG) by using antigens or
organisms
12Molecular Biology Techniques
- A- Genetic probes (DNA or RNA probes)
- Detection of a segment of DNA sequence
(gene) in unknown - organism using a labeled probe
- Probe consists of specific short sequence
of labeled single- - stranded DNA or RNA that form
strong covalently - bonded hybrid with specific
complementary strand of - nucleic acid of organism in
question - B- Polymerase chain reaction (PCR)
- Amplification of a short sequence of
target DNA or RNA Then - It is detected by a labeled probe
- C- Plasmid profile analysis
- Isolation of plasmids from bacteria and
determination of their - size and number compared with standard
strains by agarose - gel electrophoresis
13 14Bacteriophage
- Bacteriophages are viruses
- that parasitize bacterial cell
- Replication of Bacteriophage
- A- Lytic or vegetative cycle
- End by lysis of bacterial cell and release of
copies of the phage - 1) Adsorption
- Adsorption occurs between attachment sites
on the phage (tail fibres) and specific receptor
sites on bacteria - It is specific strep (sensitivity of
bacteria to different phages) -
- 2) Penetration
- The tail sheath will contract and inject
DNA into bacterial cell
15A- Lytic or Vegetative Cycle
- 3) Eclipse phase
- Viral DNA directs the host cell metabolism to
synthesize new enzymes and proteins for phage
synthesis - 4) Intracellular synthesis
- Host cell machinery is directed by genetic
information provided by phage nucleic acid to
synthesize phage coats and nucleic a. - 5) Assembly
- Protein subunits of the phage head and tail
aggregate - Each capsid acquires nucleic acid molecule to
become a mature phage particle - 6) Release
- Accumulation of huge number of phage
- The cell bursts and phage particles are
released -
16II- Temperate Phage cycle Lysogenic cycle
- Adsorption and penetration take place as in
lytic cycle - Virus DNA integrate with host chromosome
(Prophage) - and replicate as part of host chromosome
- The bacterial cell is called alysogenic
bacterium - Lysogenic bacterium has certain characters
- a- Immune to infection by another phage
- b- Acquire new properties e.g. production of
exotoxin - Diphtheria bacilli, Cl. Botulinum, Strpt.
Pyogen - erthrogenic toxin
17Outcome of Temperate cycle
- 1) The cell continue carrying prophage
indefinitely, passing it to daughter cells - 2) The prophage detach from the bacterial
chromosome and start a lytic cycle - 3) As prohage is detached it may carry genetic
material of bacterial chromosome - As it infects another bacterium , it will
transmit to it new characters
18Practical applications using phages
- Phages are important as a research tools
- Phages are used as vectors in DNA recombinant
technology - Phage typing of bacteria is important in
tracing source of infection for epidemiologic
purposes
19Antimicrobial Susceptibility testing
- Introduction
- Identification of a bacterial isolate from a
patient provides guidance in the choice of an
appropriate antibiotic for treatment - Many bacterial species are not uniformly
susceptible to a particular anti-bacterial
compound - This is particularly evident among the
Enterobacteriaceae, Staphylococcus spp., and
Pseudomonas spp. - The wide variation in susceptibility and high
frequencies of drug resistance among strains in
many bacterial species necessitates the
determination of levels of resistance or
susceptibility as a basis for the selection of
the proper antibiotic for chemotherapy
20- Antimicrobial Susceptibility testing can be down
by three ways - Minimum Inhibitory Concentration (MIC)
- Disk Diffusion Method
- Minimum Bactericidal Concentration (MBC)
211. Minimum Inhibitory Concentration (MIC)
- Principle
- The tube dilution test is the standard method for
determining levels of resistance to an
antibiotic. - Serial dilutions of the antibiotic are made in a
liquid medium which is inoculated with a
standardized number of organisms and incubated
for a prescribed time. - The lowest concentration of antibiotic preventing
appearance of turbidity is considered to be the
minimal inhibitory concentration (MIC).
22- Different concentrations of Gentamycin in
Nutrient broth - Conc. in mcg/ml
- 0.1 0.2 0.4 0.8 1.6 3.1
Gentamicin, generally considered a bacteriocidal
antibiotic, for this bacterium, has an MIC of 0.8
mcg/ml
23- Different concentrations of Tetracycline in
Nutrient broth - Conc. in mcg/ml
- 0.1 0.2 0.4 0.8 1.6 3.1 6.3 12.5
Tetracycline, generally considered a
bacteriostatic antibiotic, for this bacterium,
has an MIC of 1.6 mcg/ml
242. Disk-diffusion Method (Kirby-Bauer Method)
- The disk-diffusion method (Kirby-Bauer) is more
suitable for routine testing in a clinical
laboratory where a large number of isolates are
tested for susceptibility to numerous
antibiotics. - An agar plate is uniformly inoculated with the
test organism - A paper disk impregnated with a fixed
concentration of an antibiotic is placed on the
agar surface. - Growth of the organism and diffusion of the
antibiotic commence simultaneously resulting in a
circular zone of inhibition in which the amount
of antibiotic exceeds inhibitory concentrations.
- The diameter of the inhibition zone is a function
of the amount of drug in the disk and
susceptibility of the microorganism.
25- This test must be rigorously standardized since
zone size is also dependent on - inoculum size,
- medium composition,
- temperature of incubation,
- excess moisture and
- thickness of the agar.
- Zone diameter can be correlated with
susceptibility as measured by the dilution
method. - Further correlations using zone diameter allow
the designation of an organism as "susceptible",
"intermediate", or "resistant" to concentrations
of an antibiotic which can be attained in the
blood or other body fluids of patients requiring
chemotherapy.
26- Using a dispenser, antibiotic-impregnated disks
are placed onto the agar surface. - As the bacteria on the lawn grow, they are
inhibited to varying degrees by the antibiotic
diffusing from the disk.
27Staphylococcus aureus (MRSA)
- Note the yellowish pigmentation of the bacterial
lawn, and the lack of inhibition by the Oxacillin
disk
28Streptococcus pneumoniae (Pneumococcus)
- The brownish tint of the blood agar plate outside
the zones of bacterial inhibition is caused by
alpha-haemolysis.
29Pseudomonas aeruginosa
- The greenish tint of the lawn and plate in
general is caused by the diffusible pigment made
by the Pseudomonas aeruginosa itself.