Isolation and identification of pyogenic cocci Experiment Four Isolation and identification of pyogenic cocci

Objective of Experiment To master primary principle and method of isolation and identification pyogenic cocci from clinical specimens . To diagnose clinical disease and guide us to use medicines .

Morphologic observation 1.Staphylococci G+, arranged in grape like irregular clusters 2.Streptococci G+, arranged in chain 3.Pneumococci G+, Lancet-shaped cell. The Capsule stain: the capsule appears colorless in contrast to the blue of the cell 4.Meningococci G-, kidney-shaped ,arranged in pairs 5.Gonococci

Streptococcus Staphylococcus

Staphylococcus Streptococcus

Diplococcus

N. Gonorrhoeae N gonorrhoeae infections have a high prevalence and low mortality Acquired by sexual contact and usually affect Mucous membranes of the urethra in men Endocervix in women Infection may disseminate to a variety of tissues One of the most common STIs in the world

Morphological characteristics (Gram-stain) (Microscopic examination) PROCEDURE Isolation culture Smears (blood agar plates) Morphological characteristics (Gram-stain) (Microscopic examination) Colonial characteristics Hemolytic reaction Pigmentation Specimens morphology observation colonial Gram staining observation with the microscope Pure culture typical colonies Antibiotic susceptibility test Direct identification

Identified method for STAPHYLOCOCCI Gram-stain Isolation and culture Pure culture Direct identification The antibiotic susceptibility test

Gram-stain Morphological characteristics -Typical staphylococcus cells are spherical,arranging in irregular clusters,which just like as clusters of grape. The cell is about 1 μm in diameter. Typical cells are Gram-positive,nonmotile and do not form spore.

Staphylococci are Gram-positive cocci, typically arranged in clumps or Grape-like clusters

Isolation and culture Cultural Specimens planted on blood agar plates give rise to typical colonies in 18 hours at 370C Hemolysis and pigment production may not occur until several days later and are optimal at room temperature.

Isolation and culture colonial characteristics Colonies of staphylococci on solid meida are round,smooth,raised,and glistening.S.aureus forms gray to deep golden yellow colonies. S.epidermidis colonies are gray to white on primary isolation. Various degrees of hemolysis are produced by S.auerus and occasionally by other species.

Pure culture MATERIALS: PROCEDURE . Agar slope culture Colonies on agar plate PROCEDURE With the flame-sterilized wire inoculating loop, transfer a small amount of bacteria from the colony on agar plate. Then streak on the agar slope. Sterile the mouth of tubes, replug the test tubes and flame the loop. Label and incubate at 37℃ for 18-24 hours .

Directed identification The Coagulase Test The Dnase Test The mannitol fermentation test. The phage typing test Animal Experiment

The Coagulase Test Possession of the enzyme coagulase which coagulase plasma is an almost exclusive property of Staph.aureus.There are two ways of performing this test The Slide Coagulase Test The Tube coagulase Test

The Coagulase Test Coagulase is an enzyme converting fibrinogen into fibrin promoting blood clotting. It has been speculated that this enzyme might be a virulence factor with the coagulated blood around the bacteria protecting them from the immune system. However, coagulase-negative strains are often as pathogenic as coagulase-positive strains.

The Slide Coagulase Test

The Tube coagulase Test positive negative

The Dnase Test Inoculate Dnase agar plates with a loop so that the growth is in plaques about 1 cm in diameter.Incubate at 370C overnight.Flood the plate with 1 N hydrochloric acid.Clearing around the colonies indicates Dnase activity.The hydrochloric acid reacts with unchanged deoxyribonucleic acid to give a cloudy precipitate.A few other bacteria,e.g. Serratia,may give a positive reaction.

The mannitol fermentation test. Inoculate the bacteria into a mannitol micro-tube,incubate at 370C for 18h.S.aureus will ferment mannitol to produce acid,which causes the medium to turn yellow.

The phage typing test This test is used to trace the infective agent in epidemiology if necessary.It is usually not done for routine clinical purpose.

Animal Experiment Isolation and identification of bacteria Vomit excrement remaindered food observation filter Meat soup media 6--8W cat food poisoning injection

The antibiotic susceptibility test This test is helpful for the treatment of S.aureus infection. materials S.aureus（isolated from the pus of a patient）. Several kinds of filter paper （each contains different kinds of antibiotics） Nutrient agar plate

The antibiotic susceptibility test PROCEDURE Streaking the S.aureus on agar plate (thoroughly covered the plate) Put 4 kinds of paper contained different antibiotics on the plate (each paper are far away about 2 cm) Incubate ar 370C 18-24 hours. Observe the results the plates are examined for the present of zones of inhibition of bacterial growth around the filter paper.The sensitivity of the organism is indicated by the diameter of the zone of growth inhibition.

Hiss’ stain Dissection of white mice Materials Animals:mice died from infection Instruments for autopsy:sterile tweezers and scissors,anatomic board Pins,iodine,ethanol,cotton absorbent gauze balls,3%lysol ,and so on

How to make a smear Open the abdomen cavity from pubis and expose the abdominal viscera . Check the intestine Put tissue fluid on the slide

Examine under the oil immersion lens Hiss capsule staining 1%crystal violet in alcohol Smear Dry 2mins Purple black cells of bacteria and colorless capsule Wash with 20%Cuso4 Examine under the oil immersion lens Blot dry with bibulous paper

Pneumococcus