Practicals WT4 Mycobacterium,
Corynebacterium
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Laboratory diagonosis of mycobacteria + ATB susceptibility tests
Laboratory diagnosis of. corynebacterium + ATB susceptibility test
Lysogenic conversion
Antituberculotics
Microscopy - M.tbc - Ziehl Neelsen, C, diphtheria - Gram, Albert, C.
pseudodiphthericum - Gram
• Cultivation - M. tbc - Lowenstein and broth medium, C. diphtheriae a
C. pseudodiphthericum - Blood agar and tellurit medium
• Biochemical characteristics of C.diphtheriae a C. pseudodiphthericum
• Toxigenicity of C. diphtheria - ELEK´s method and animal model
Laboratory diagnosis of
mycobacterium
• Sampling - in pulmonar tbc - morning sputum -3 consecutive
days, extrapulmonar - appropriate sample - big quantity of liquid urine, menstrual blood…
• Microscopy - Ziehl Neelsen, fluorescence - increased sensitivity,
preliminary dg
• Cultivation -sample proceeding, decontamination of
nonmycobacterial flora, condensation of the sample - NaOH,
centrifugation. Eggs media - Šula, Lowenstein / solide and broth /
- pigment, shape - in broth - membrane, sediment - Long
generation time - quick methods - based on detection of
metabolits of palmit acid and CO2
• Genetic probes - detection of aminoacids sequences
Staining
• Ziehl Neelsen - cover heat fixed and dried smear
with a piece of filtrate papaer. Apply 5-7 drops of
carbolfuchsine. Heat from beneath till vaporisation. Discard
paper with forceps, rinse and dry. Decolorise with acid
alcohol (HCl+ethanol) untill the color is stining the water.
Conterstain with methylen blue. Rinse, dry. M. tbc - red,
surroundings - light blue.
• Albert - methylen blue or toluidin blue
Lab. dg. of corynebacterium
• Diagnosis of the disease is based on clinical picture and
epidemiological history. Lab. dg. is long and complicated
Appropriate sampling
• Microscopy - smear with Gram staining and Albert´s staining does
not distinguish between C. diphtheriae and other Corynebacteria.
• Cultivation: Blood agar - 3 typs od colonies - mitis, gravis,
intermedius, Loffler´s medium, tellurit medium brown colonies with
halo
• Biochemical identification- differentiation from other corynebacteria
present in throat
• toxigenicity of the strain - ELEK,
• ATB susceptibility
Antituberculotics and therapy
• Streptomycin(kill actively multiplying M.tbc)
Izoniazid,Rifampicin (effective on M.tbc in caseous
necrosis) Ethanbutol Etionamid Kanamycin Cycloserin
Pyrazinamid ( active i.c.)
• In pacient with pulmonar tbc - 3 populations of M.tbc
- localised extracelullarly in cavitas,
intracelullarly in macrophages,
inside caseouse necrosis
• Combination of 2-3 ATT. Resistence fighting -overgrowinf
of resistent mutants in culture of M.tbc - for INH 1:105 for
STM 1:106 in infectious place there is 107- 10 9 of bacteria
Lysogenic conversion
• Strain of C. diphtheriae gains his
toxigenicity by lysogenic conversion thanks to bacterophage. Bacterial virus bactrophage is able to transfere genetic
information about the production of toxin tox gen - fom one cell ( toxigenic) to the
other (non toxigenic) C. diphtheriae cell.
Cultivation of Corynebacteria
• C. diphtheriae - blood agar
• C. pseudodiphthericum - blood agar, inverse
CAMP
• C. diphtheriae on tellurit medium - brown
colonies with brouwn ring - halo
Microscopy
• Ziehl Neelsen staining - acid-fast M. tbc - not willin
to accept stain. If colored - very difficult to
decolorise
• Gram staining of C. diphtheria - G+rods
• Albert´s staining - metachromatic staininig of
granules - resulting color is different from color used
for staining - C. diphtheriae
• Comparison of Gram smear of C. diphtheriae a C.
pseudodiphthericum
Cultivation of M. tbc
• Egg medium - Lowenstein - colony
morphology, no pigment
• Broth medium - growing in membrane
• cultivation 3-6 weaks - reading every weak
Biochemical characteristics
• Fermentation: differentiation of C. diphtheriae from
other corynebacteria isolated from throat
(C.ulcerans, C. pseudotuberculosis, C. xerosis, C.
pseudodiphthericum
• C. diphtheriae
• granul catalase gelatine, urea, lactose, maltose, glc
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• C. pseudodiphthericum
Detection of toxigenicity of C.
diphtheriae
• ELEK´s test of toxigenicity - imunodiffusion of
suspension of tested strain and antidiphtheric serum
in agar - zone of precipitation
• Annimal mode: application of
diphtheria toxin i.d. ………………..necrosis
• antidiphtheric serum (i.p. or i.d.) + toxin …no
necrosis
ATB susceptibility
• Susceptibility test for antituberculotics - ATT are dissolved
in medium
• Method of proportional susceptibility testing - dilution
method. Dilution of inoculum so that there is from 100-300
CFU - colony forming units on a plate
• If there is more than 1% of M. tbc cells resistent to ATT
the therapy will not be cliniccňally suscessful
• Susceptibility of Corynebacterium - PNC, ERY - for
elimination of bacteria or carriage state - therapy of
diphtheria - primarily antitoxic. Preventive measures.
• Nondiphtheriae corynebactria - Vancomycin, (Cefalosporins
of the 1st generation)
• Spontaneous appearance of resistent mycobacteria without exposition to ATT
• Frequency of resistent cells in the culture is 1:105 for
INH and 1:106 for STM - if both are given in
combination the incidence of resistent strains will be
1:1011.
• The overall population of bacilli in patient with open
cavity is 10 7 - 109 , so there is 102 - 104 resistent strains.
• 1 ATT means overgrowing of resistent strains that will
replace susceptible killed bacteria.
• Combination of 2 and more ATT: INH+RIF - 9 months,
+ STR+ Ethanbutol for 2-8 weaks, from 2nd months
only 2 times weakly, INH + Ethanbutol 18-24 months,
• Tests for ATT susceptibility - correlation between in
vitro and therapy success.
• If more than 1% of M. tbc in vitro are resistent - therapy
will not be successful
• Tests determine ratio between susceptible and resistent
strains
• Agar dilution method - depend on innoculum - if too
thick - there is too much of resistent strains - false
resistent, if less concentrated - false positive - possible
resistent strains not identified.
• Optimal dilution 100-300 CFU on plate
• Inconvenience - aga can inhibit activity of ATT as well
as of mycobacteria, long incubation decrease activity of
ATT
Principles of therapy
• Therapy: ATT are active only on growing
mycobacteria, growth depends on oxygen availability
and pH (optimu neutral or alkaline) - Max in open
cavity, min in caseouse necrose.
• In i.c. - in phagosomes - pH is 5,5, acid - slow
growth, less of bacilli, less resistent strains - ATT
supporting acid environment - Pyrazinamide. (STM
not able to enter phagosomes and looses activity in
acid pH)
• in chronic closed caseouse necrose - slow blood
supply and oxygen, slow metabolism, less living
cells, pH neutral but slow multiplication
ATT
• Bactericidal: STM - mycobacteria extracelullarly in
cavities
• INH - kills slowly and quickly growing cells
• Rifampicin - effective on mycob. Closed in caseouse
necrosis and macrophages
• Pyrazinamide - only in acid pH and in macrophages
• Bacteristatic: Ethanbutol - only in combination, able to
penetrate in mycobacteria situated i.c. and e.c.
• Capreomycin, Kanamycin - bactericidal, for e.c
mycobacteria in cavities
• Ethionamid, cycloserin - bacteriostatic for i.c and e.c