Gram staining Techniques

Some history Bacteria are translucent Bacteria are translucent Staining make them visible under microscope Staining make them visible under microscope 1884 Hans Christian Gram  stained cells and found that some lost their color when excess stain was washed off 1884 Hans Christian Gram  stained cells and found that some lost their color when excess stain was washed off Differential stain  distinction between 2 types of bacteria Differential stain  distinction between 2 types of bacteria

Bacteria cell walls Peptidoglycan: network of sugars cross- linked by short peptides Peptidoglycan: network of sugars cross- linked by short peptides Forms the rigid part of the cell wall Forms the rigid part of the cell wall Protects the bacteria against mechanical damage Protects the bacteria against mechanical damage Part that picks up the stain in the gram procedure Part that picks up the stain in the gram procedure www-micro.msb.le.ac.uk/ video/graphics/wall.gif

Gram - bacteria Small peptidoglycan Small peptidoglycan Are stained with crystal violet but decolorized with alcohol after which they pick up the red stain Are stained with crystal violet but decolorized with alcohol after which they pick up the red stain LPS on outer membrane  toxic for host LPS on outer membrane  toxic for host

Gram + bacteria Large, highly cross- linked petidoglycan Large, highly cross- linked petidoglycan No outer membrane No outer membrane

gsbs.utmb.edu/microbook/ images/fig2_6.jpg

Bacteria shape Long rod: bacillus Long rod: bacillus Round shaped: cocci Round shaped: cocci Spiral: Spirochete Spiral: Spirochete

Bacteria shapes

Some bacteria shapes spirochete cocci coccobacillus bacillus

Gram stain Is the most commonly used technique to stain bacteria Is the most commonly used technique to stain bacteria Almost always first step into identification Almost always first step into identification Tell what other tests to perform Tell what other tests to perform Gram – bacteria stain red-pink Gram – bacteria stain red-pink Gram + bacteria stain blue-purple Gram + bacteria stain blue-purple

Gram Positive Bacteria (blue/purple) Gram Negative Bacteria (Red/pink)

Gram stain recipe Smear Smear Heat fix Heat fix Crystal violet (30 sec)  wash Crystal violet (30 sec)  wash Iodine (1 minute)  wash Iodine (1 minute)  wash Ethanol (varies ~15 sec)  wash Ethanol (varies ~15 sec)  wash Safranin (30 seconds)  wash Safranin (30 seconds)  wash Blot dry Blot dry

How to prepare a smear Label your slide with a pencil Label your slide with a pencil Use a clean slide: manipulate by the edges Use a clean slide: manipulate by the edges Using a sterilized loop spread of drop of bacterial culture on the slide Using a sterilized loop spread of drop of bacterial culture on the slide If solid culture  put a drop of water on the slide  add a little bit of bacteria (using loop) If solid culture  put a drop of water on the slide  add a little bit of bacteria (using loop) A thin film is better A thin film is better Dry faster, distribution of dye and decolorizer more even Dry faster, distribution of dye and decolorizer more even No coverslip on! let dry (until most water evaporated) No coverslip on! let dry (until most water evaporated)

Fixation Pass the slide through the flame of the bunsen burner Pass the slide through the flame of the bunsen burner Moving in a circular motion (to avoid localized overheating) Moving in a circular motion (to avoid localized overheating) The slide should not be too hot to touch, slightly warm but no more! The slide should not be too hot to touch, slightly warm but no more! If too hot  overfixed  burn bacteria everything will be black If too hot  overfixed  burn bacteria everything will be black If underfixed  bacteria do not stick to slide and will be washed away during the staining procedure If underfixed  bacteria do not stick to slide and will be washed away during the staining procedure

Why bacteria need to be fixed Denature bacterial enzyme  prevent them from digesting the cell (autolysis) Denature bacterial enzyme  prevent them from digesting the cell (autolysis) Make the bacteria stick to the slide so they are not washed away during staining. Make the bacteria stick to the slide so they are not washed away during staining.

1st stain Crystal violet Crystal violet Colorize all cells Colorize all cells Flood the slide with crystal violet Flood the slide with crystal violet ***make sure that the bacteria are on top ***make sure that the bacteria are on top 30 seconds 30 seconds Rinse gently with running tap (or distilled) water Rinse gently with running tap (or distilled) water Do not squirt water directly onto the smear Do not squirt water directly onto the smear Shake off the excess water Shake off the excess water

Mordant Iodine Iodine Make the dye stick to the cell wall Make the dye stick to the cell wall Crystallize the dye in the peptidoglycan Crystallize the dye in the peptidoglycan Flood the slide for 1 minute Flood the slide for 1 minute Wash Wash

Decolorization Ethanol seconds, until dye doesn’t run out anymore Ethanol seconds, until dye doesn’t run out anymore Critical step***** Washing after is very important (stop alcohol action) Critical step***** Washing after is very important (stop alcohol action) Cell that have thin peptidoglycan decolorize Cell that have thin peptidoglycan decolorize Long story: Long story: Dissolve the lipid layer from the gram negative bacteria Dissolve the lipid layer from the gram negative bacteria Enhance the leaching from the primary stain Enhance the leaching from the primary stain In gram + bacteria: alcohol dehydrate the thicker cell wall In gram + bacteria: alcohol dehydrate the thicker cell wall Prevent diffusion of the violet iodine complex Prevent diffusion of the violet iodine complex

Ethanol Sink

Counter stain Safranin Safranin  color pink  color pink Flood the slide for 30 seconds Flood the slide for 30 seconds Rinse gently with water and shake off the excess water from surface Rinse gently with water and shake off the excess water from surface

Blotting Slides can be air dried or blotted Slides can be air dried or blotted Blotting  put between 2 sheets of absorbent paper (we will use bibulous paper). Blotting  put between 2 sheets of absorbent paper (we will use bibulous paper). DO NOT RUB THE SMEAR (bacteria will come off)…just blot between papers. DO NOT RUB THE SMEAR (bacteria will come off)…just blot between papers.

Oil immersion Light is refracted when goes trough Light is refracted when goes trough slide (change in media from glass to air) Oil has same refractive index than glass  light ray goes with no refraction Oil has same refractive index than glass  light ray goes with no refraction Use only one drop of oil Use only one drop of oil Only use the 40X objective (greatest power objective) with oil immersion. Only use the 40X objective (greatest power objective) with oil immersion. Clean the objective with paper lens after use Clean the objective with paper lens after use

Oil immersion

Gram Staining Video HHNZ8cdo HHNZ8cdo HHNZ8cdo HHNZ8cdo media/Instructional.Stain.Gram.php media/Instructional.Stain.Gram.php media/Instructional.Stain.Gram.php media/Instructional.Stain.Gram.php

Objectives for Tomorrow’s Lab You have 2 bacteria You have 2 bacteria S. Urea S. Urea P. fluorescens P. fluorescens For each bacteria: Stain each bacteria Stain each bacteria Observe and identify the bacteria shape and gram results. Observe and identify the bacteria shape and gram results. You are doing this lab in team but make sure that each student perform at least 1 gram stain You are doing this lab in team but make sure that each student perform at least 1 gram stain

Resources 205/lab/Lab_3_Gram_stain_spring_07. ppt 205/lab/Lab_3_Gram_stain_spring_07. ppt