MICROBIOLOGY

MB102b

LABREPORT#3:

NegativestainingofStaphylococcusaureuswith
nigrosin

MuhammedSayedHassan
ID:171741
GROUP(F)

Submittedto:L.A.Suzanneali

NegativestainingofStaphylococcusaureuswith
nigrosin

INTRODUCTION

Thislabcoveredsmearpreparationofstaphylococcusaureusonthe
microscopicslidewiththeheatfixationmethod,Thennegativestainingwith
acidicdye(Nigrosin)andsuccessfullyidentifyingit'sshapeunderthelight
microscope.Thenegativestainingmethoddependsontherepulsionofthe
bacterialcellsurfaceofthenegativechargeddyetocolorizethe
background(microscopicslide).Aimofthelabistonegativestainstaphylococcus
aureusandidentifyit'sshapeunderthelightmicroscope.

MATERIALS

Chemicalagents:

1. Immersionoil
2. Nigrosin(Nigrosin100gm/L,Formalin5ml/Linwater)
3. Distilledwater
4. Ethanol(70%)

Biologicalagents:

1. Staphylococcusaureus

Equipments:

1. LightMicroscope
2. Microscopicslide
3. Aluminiumfoilsheet
4. Inoculationloop
5. Bunsenburner

PROCEDURE

a) Smearingofbacteria
1. 1dropsofwaterwasputonthemicroscopicslide.
2. Theinoculationloopwassterilizedbybunsenburner.
3. 1loopfulwasputonthemicroscopicslide.
4. Thebacteriawassmearedontheslide.

b) Heatfixationofbacteria
5. Thebacteriawasheatedwithbunsenburnerwithindirectflamefixation.

c) Negativestainingoftheslide
6. Themicroscopicslidewasputonthealuminiumfoilsheet.
7. 2dropofdyeweredroppedontheedgeofthemicroscopicslide.
8. Anotherslidewasusedatangleof45tosmearthedyecarefullyonthebacteria
slide.
9. Thentheslidewasdroughtforabout10minutesbeforeexamination.

d) Microscopicexaminationoftheslide
10. 1dropofimmersionoilwasdroppedontheslide.
11. Theslidewasputinthemicroscopicclip.
12. Knobwaszoomeduntilthelenstouchedtheoilconcavesurface.
13. ThentheslidewasexaminedwiththeOillens.

RESULTS

Fig(1): Negative stain of staphylococcus aureus bacteria shown under a light microscope
Using Oil-immersion technique.

DISCUSSION

Abovefig(1)showsthatculturedstaphylococcusaureusunderlightmicroscopeusing
oil-immersionlens.Smearandnegativestainingwassuccessfullyapplied,the
backgroundoftheslidehadthedyebutthebacterialcellsurfacerepulsedthedye
becauseit'sacidicandhasanegativechargesasthesurfaceofthebacterialcell.

CONCLUSION

Theaimofthelapwassuccessfullyachievedwhichisnegativestainingofstaphylococcus
aureususingnigrosinacidicdye.