Gram stain - Fudan Universityfdjpkc.fudan.edu.cn/_upload/article/files/de/01/4e502ce...Gram stain 1. Cover the smear with for 2. Briefly wash off the stain. Drain off excess water

Experiment 1Experiment 1

Gram stainGram stain

Yuyan Wang

MEDICAL MICROBIOLOGY EXPERIMENT 1 Wang Yuyan

Bacterial stain

Living bacteria stain

Killed bacteria stain

Positive staining Negative staining

Common stain

Single-dye stain

Special stain

Complex stain Endospore stain Capsule stain

Gram stain

Cell wall stain

Acid fast stain

Methods for staining bacteriaMethods for staining bacteria


Demonstation Demonstation

Escherichia coli (Gram stain, oil 100 )

100 )

×

大肠埃希菌（革兰染色，油镜 ×


Streptococcus (Gram stain, oil 100 )

100 )

×

链球菌（革兰染色，油镜 ×


StaphylococcusStaphylococcus (Gram stain, oil 100 )

100 )

×

葡萄球菌（革兰染色，油镜 ×


Neisseria meningitidis (cerebrospinal fluid methylene blue stain, oil 100 )

100 )

， ×

脑膜炎双球菌（脑脊液，美兰染色，油镜 ×


Capsule of pneumococcuspneumococcus capsule stain, oil 100 )

100 )

（ ×

肺炎球菌的荚膜（荚膜染色，油镜 ×


Spore of spore stain, oil 100 )

100 )

Clostridium tetani （ ×

破伤风梭菌（芽胞染色，油镜 ×


Flagella of Bacillus proteus（ ×

变形杆菌的鞭毛（鞭毛染色，油镜 ×

Flagella stain, oil 100 )

100 )


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Thick peptidoglycan90% peptidoglycanTeichoic acidsNot many polysaccharides

Gram stain

Divides bacteria into 2 groupsDivides bacteria into 2 groups

GramGram--positivepositive GramGram--negative negative

Thin peptidoglycan

5-10% peptidoglycan

No teichoic acids

Outer membrane has lipids, polysaccharides


applicationapplication

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Identification:

Color, shape, arrangement, endospore

Diagnosis

toxin, endotoxin

Treatment

antibiotic selection: G+, G-


primary stain: crystal violet

Mordant: Gram’s iodine

Decolorization 95% ethanol

Counterstain: safranin

Effects of reagents on bacterial cells

, causes both gram-positive and gram-negative organisms to become purple.

combine with crystal violet to form a relatively insoluble compound in the bacteria.

: , the gram-negative bacteria are leached colorless.

, adds a pink color to the decolorized gram-negative bacteria without affecting the color of the purple gram-positive bacteria


Materials Materials l

l

l

4 students sharing

2 students sharing

Everyone:

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Cleaning paper for lens (the blue box) 1 bottle of immersion oil (cedar oil and aether (cleaning solution)1 bottle of salineDrying paperMarker

Bacteria Strains:and (cultures on slants).

Gram stain kit:1Bunsen burnerDipper

Microscope slides: 1-2Inoculating loops:1Microscope (in the storages under the bench)

）

Staphylococcus albus Escherichia coli


Gram stain Gram stain

® Procedure1. Prepare bacterial smears

2. Gram stain

3. Observation by microscope


Preparation of smearPreparation of smear

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Take a clean slideMaker the slide Flame an inoculating loop, let it cool, and transfer two loopfuls of water to the center of the target circle.

Strain 1 strain 1 + 2 Strain 2

Your slide may be dived into three areas for staining


CAUTION !

Pick up a verysmall amount ofthe organisms

Pick up organisms from a tube slant

Be sure to cool the loop completelybefore inserting it into a medium. A loopthat is too hot will spatter the mediumand move bacteria into the air.


Making bacterial smears

Be sure to flame theinoculating loop beforereturned it to rack.



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Gram stainGram stain1. Cover the smear with for 2. Briefly wash off the stain. Drain off excess water.3. Cover the smear with solution for 10 seconds.4. Pour off the Gram s iodine and washed by water.5 Flood the smear with

Thick smears will require more time than thin ones.

6. Stop action of the alcohol by rinsing the slide with water for a

7. Cover the smear with 8. Wash gently for a few seconds, blot dry with bibulous paper, and

air-dry.9. Examine the slide under oil immersion.

crystal violet 10 seconds.

Gram s iodine

95% ethyl alcohol for This step is critical.

safranin for

’10 to 20 seconds.

Decolorization has occurred when the solvent flows colorlessly from the slide.

10 seconds.

few seconds.


10 10 seconds

10





OilOil ImmersionImmersion

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Turn on microscopeAdjust brightnessInsert slideAdd drop of oil on TOP OF COVERSLIPRotate nosepiece until 100X objectives straddle slideRotate oil immersion lens (100x) into oilUse coarse adjustment to lower stage until specimen appearsUse fine adjustment knob to sharp focusObserve and draw

Clean objectives with lens paper when finished!



Report Report Requires1. Objective 2. Principles 3. Materials 4. Procedure5. Results (draw what you observed by the microscope,

or describe the phenomenon or changes emerged in your experiment )

6. Discussion (the notes in procedures or analysis of the results)


Questions Questions

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Which step is the most crucial or most likely to cause poor results in the Gram stain? Why?

Which phase of the bacteria ther have a typical shape and color by Gram staining, lag, l ? why?

What part of the bacterial cell is most involved with Gram staining, and why?

How to protect yourself against lab-infection in the medical microbiology lab (BSL-1,2).

ogarithmic, stationary, decline phase

Documents

Gram stain - Fudan Universityfdjpkc.fudan.edu.cn/_upload/article/files/de/01/4e502ce...Gram stain 1. Cover the smear with for 2. Briefly wash off the stain. Drain off excess water