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Practical 7
Biochemical properties testing
1 part
Primary innoculation on cultivation media according to
the sample:
basical media (blood agar) and 1 selective or diagnostic medium –
shorter diagnostic process
Blood agar + chocolate agar (Respiratory tract ,CNS – for Haemophilus
sp)
+ McConkey (stool,urine, clinical materials (G-rods)
Primary – 1st day isolation – basical orientation
according to morphology of colonies, Gramm staining
- decission for next procedures
- orientation quick tests – (catalase, cytochromoxidase, bile solution testi,
slide coagulase)
Subcultures
- multiplication of one colony and performance of differential test based
on biochemical properties
Cultivation media
- basical media – microbes without special nutrition requirements, multiplication –
(bouillon, meat extract, pepton broth)
-enriched media – basical media + enrichment factor (hemoglobin, fildes extract, yeast
extract, chemically defined enrichment factors)
-diagnostic media – detection of some biochemical properties of bacteriaí – sugar
medium for biochemical activity of feremtation of ´glycides,- decaroxylation of
aminoacides, production of some molecules during metabolism (H2S). These media
contain the indicators. If bacterium is metabolising the structure in the medium acid is
formed and pH changes – decrease – and the color indicator change also – visualisation
of chemical reaction
-selective – for growth only of selected bacterium (medium with special growth factors
or inhibitors of growth (egg media, ATB media, Kauffman medium Campylobacter
medium, Legionella, mycoplasma medium .....
-selektively diagnostic – base + inhibitors + substrate of metabolism + indicator Endo,
MacConkey, DC agare – G-paličky Lactose negative or positive
-media for storage, transport, ATB susceptibility testing
Tests for biochemical properties and metabolic activity
testing
Aim – final identification
Method – subcultivation on the series of testing diagnostic meida
Liquid media – with chemical structure – substrate and indicator, solid
media with biochemical – metabolic substrate and indicator and
diagnostic disc with substrate, micromethods – liquid media with
substrate and indicator in microwells
Algorithm – of chosen procedures
Group of biochemical tests aligned so that they allow numeric
identification based on statistical probability of the result of one test In
the positive result the well is attributed the chifer according to the
position in the triplet.( 1 2 or 4) Addition of chifers in triplet gives the
number and each result of the triplet gives a subsequent one position of
the code that is the combination of numbers of tested triplets. This code
is corresponding to one bacteria
--- 0
+-+5
-++ 6
+ + -3
+-- 1
+++ 7
-+- 2
--+ 4
Demonstration
G+cocci: Staphylococcus aureus a Staphylococcus epidermidis on the
selective-diagnostic medium Salt mannit:
selectively NaCl allowed growing of staphylococci that tolerate
it.while others do not., mannitol is the diagnostic substrate utilised by
St. aureus which metabolised it, formed acid that makes the medium
becomming acid and change the pH and indicator color. St. aureus
changes the original red color to yellow, St. epidermidis is growing on
the mediu , tolerates salt without changing the pH and indicator color not utilising manitl
G-rods
Hajn
glucose succrose lactose
maltose manit indol urea/motility
H2S
Proteus vulgaris
+
+
-
+
-*
-
+
+/+
Proteus mirabilis +
+
-
-
-
-
-
+/+
Salmonella typhi +
+
-
-
+
+
-
-/+
Escherichia coli
-
+
+
+
+
+
+
-/+
Shigella
-
-
-
-
-
+
+
-/+
Yeast – zymogram, fermentation of glycides – biochemical properties –
diagnostic medium: glc,sach,mal,lak,raf +/oranžová, -/modrozelená
–––––––––––––––- + + - Helicobacter pylorí
fermentation of urea, Helicobacter has urease activity that hydrolyse urea
(making so a good environment– NH4 – for surviving in acidic stomach
Surface of tube medium – aerobe environment, lactose negative bacteria do not ferment, it is
alkaline, red
Lower part - in anaerobe environment, enterobacteria ferments glycids – acidic – yellow or black
H2S .
1 negative control 2 Ps.aeruginosa : net fermenting - red
3 Shigella sonnei: H2S - negat.,gas – negat., TSI – acid/alcalic red/yellow
4 Salmonella typhi: H2S – pozit., gas–negat., TSI – acidic/alcalic red/yellow
5 Escherichia coli: H2S – negat.,gas -posit., TSI – acid/acidic
red/red
6 Proteus mirabilis: H2S – posit, gas - negat., TSI – acid /acid red/red
Klin
Combined diagnostic medium solidified in bent position of Petri
dishes, in formed of triangle to 1/3 of the dishes. Endo medium is
added too. The medium is thickly innoculated together with some ticks.
The cover glass is put - on the surface - for the detection of gas.
Fermentation of glc, urea, thiosulfate is detected. Bacteria that
hydrolyses urea forme NH4 what change pH and the color of green
indicator to blue. Fermentation of glc changes the color of the triangle
to yellow, formation of gas that is seen under the cover glass.
Producers of H2S turn the medium black.
Escherichia coli glc +, gas +, H2S-
Citrobacter glc+, gas +, H2S+
Proteus gas-, H2S+
MikroLaTest
Identification of Klebsiella pneumoniae – Enterotest
+ /- + - - +- + +
+ + + + + + + + /+
Detection of catalase and oxidase activity
Catalase: - enzyme, hydrolysing H2O2 – toxic for the cell and formation
of molecular oxygen. Moraxella catarrhalis –cat.negat.
H2O2 hydrolysis, bubbles - Staphylococcus sp.
Oxidase: - enzyme active in end phases of metabolism of strict aerobes
Pseudomonas aeruginosa – colonies turn black after drop of
dimetylparaphenylendiamine is added– (Neisseria gonorrhoe)