Gram Staining & Microscopy

This is the procedure you'll follow at the bench. Open a part to read it step by step, with the reason for each step, before you start.

1. 1

   Gown up5 steps, in order

   Choose the right protection for the work and put it on in the right order.

   The method for this part

   1. 1

      Tie hair back, remove jewellery

      First, before your hands are washed: you won't touch your head again once they're clean.

   2. 2

      Lab coat, fastened

      The coat goes on before you wash, so clean hands don't have to handle it, and before gloves, so the gloves can cover its cuffs.

   3. 3

      Wash your hands

      Hands are washed once your hair and the coat are dealt with, and before you touch clean eye protection and gloves. With rings and watch off, the whole hand and wrist can be reached.

   4. 4

      Safety glasses

      Eye protection goes on before gloves, with freshly washed hands.

   5. 5

      Nitrile gloves

      Gloves go on last, over the cuffs and onto clean, dry hands, so they're clean when you start work.

   What goes wrong here

   • Respirator. Routine bench work doesn't need a respirator. Wearing more than the risk calls for just makes the work clumsier.
   • Plastic kitchen apron. A kitchen apron leaves your arms bare. In a lab you need a fastened coat with sleeves.
   • Your own jacket. Outdoor clothes stay in the locker: they carry contamination in and out.
2. 2

   Collect your kit12 things to fetch

   Fetch what the test needs from the stores around the lab, or have it brought to you.

   The method for this part

   What you check

   • • Glass slides
   • • Marker pen
   • • Inoculating loop
   • • Bunsen burner
   • • Broth culture
   • • Crystal violet
   • • Gram's iodine
   • • Decolouriser
   • • Safranin
   • • Deionised water
   • • Immersion oil
   • • Lens tissue

   What you decide by

   • • Take only what the test uses: anything extra on the bench is clutter to reach over.
3. 3

   Prepare the smearBench work

   Label the slide, make a thin smear with controls, air-dry and heat-fix it.

   The method for this part

   1. 1

      Label the frosted end of the slide.

      An unlabelled slide can be mixed up with someone else's.

   2. 2

      Flame the loop until it glows red, then let it cool.

      A red-hot loop is sterile, so only your isolate goes onto the slide.

   3. 3

      Put a small drop of saline in the middle of the slide.

      Cells from a colony need a little liquid to spread into a thin film.

   4. 4

      Touch one isolated colony lightly with the loop.

      An isolated colony grew from a single cell, so it's pure. You need only a barely visible amount.

   5. 5

      Mix it into the drop and spread a thin film about the size of a fingernail.

      Cells must lie in a single layer to show their shape and arrangement.

   6. 6

      Flame the loop again.

      It's carrying live culture: sterilise it before it touches anything else.

   7. 7

      Add control smears: Staphylococcus (Gram-positive) and E. coli (Gram-negative).

      Controls of known organisms prove whether the stain worked on this slide.

   8. 8

      Flame the loop once more before putting it down.

      Never put down a loop that still carries culture.

   9. 9

      Let the smears air-dry completely.

      A wet smear boils in the flame, spattering and distorting the cells.

   10. 10

       Pass the slide through the flame 2–3 times, smear side up.

       Gentle heat sticks the cells to the glass so they survive the rinses.

   What goes wrong here

   • Taking from the crowded part of the streak. Colonies there touch each other, so two organisms can come up on one loop. Take a single colony standing on its own.
   • Scooping up a whole colony. Too much makes a thick smear, where the cells pile up and can't be read.
   • Heat-fixing a smear that is still wet. It boils in the flame, which spatters live cells and distorts the ones left.
4. 4

   Stain4 timed reagents

   Crystal violet, iodine, decolouriser and safranin, each for the right time, rinsing between.

   The method for this part

   1. 1

      Flood the slide with crystal violet.

      The primary stain soaks into every cell and turns it violet.

   2. 2

      Leave it 45–90 s, then rinse gently with water.

      The rinse stops the reagent and washes off the excess.

   3. 3

      Flood the slide with gram's iodine.

      The mordant binds crystal violet into a large complex inside the cell.

   4. 4

      Leave it 45–90 s, then rinse gently with water.

      The rinse stops the reagent and washes off the excess.

   5. 5

      Add acetone-alcohol drop by drop, watching the run-off.

      Acetone-alcohol washes the complex out of thin Gram-negative walls but not thick Gram-positive ones.

   6. 6

      Rinse as soon as the run-off is clear (5–20 s).

      The critical step: too long and Gram-positive cells lose their violet.

   7. 7

      Flood the slide with safranin.

      The counterstain turns the now-colourless Gram-negative cells pink.

   8. 8

      Leave it 30–90 s, then rinse gently with water.

      The rinse stops the reagent and washes off the excess.

   9. 9

      Blot the slide dry with bibulous paper.

      Press gently; wiping across the smear would rub the cells off.

   What goes wrong here

   • Leaving the decolouriser on too long. Gram-positive cells lose their violet and come out pink: a wrong answer that looks like a right one.
   • Wiping the slide dry. Wiping rubs the smear off the glass. Blot it.
5. 5

   MicroscopyMicroscope

   Focus up through the objectives to oil immersion and read the cells.

   The method for this part

   1. 1

      Swing in the 10× objective.

      Low power shows you where the smears are before you zoom in.

   2. 2

      Raise the stage with the coarse focus until the smear comes into focus.

      Focusing at low power first means you're nearly in focus at high power.

   3. 3

      Add one drop of immersion oil onto the smear.

      Oil matches the glass, so the 100× lens can resolve bacteria.

   4. 4

      Swing in the 100× oil-immersion objective.

      Bacteria are only 1–3 µm long: you need 1000× magnification.

   5. 5

      Bring the cells into sharp focus with the fine focus only.

      Never use coarse focus at high power: the lens could hit the slide.

   6. 6

      Turn the light up until the image is bright but the colours are still strong.

      Oil immersion needs plenty of light; too much washes the colours out.

   7. 7

      Move to each control smear in turn, and check its colour.

      Read the controls first: they tell you if this stain can be trusted.

   8. 8

      Now move to a thin area near the edge of the isolate smear, where cells lie in a single layer.

      The thick centre piles cells up; the feathered edge shows true shape and grouping.

   What goes wrong here

   • Starting at high power. The smear can't be found at 100×. Find it at 10× first, where there is room to look.
   • Using the coarse focus at high power. It can drive the lens into the slide, cracking both.
   • Getting oil on the 40× lens. It is a dry lens: oil ruins its image, and the lens.
6. 6

   ReportLab report

   Check the controls, then report Gram reaction, shape and arrangement.

   The method for this part

   What you check

   • • The Gram-positive control: violet
   • • The Gram-negative control: pink
   • • The isolate, where its cells lie one deep

   What you decide by

   • • If either control is the wrong colour, the stain has failed: report that it must be repeated, and nothing about the isolate.
   • • If both controls are right, report the isolate's Gram reaction, the shape of its cells, how they are arranged, and whether it is one kind of cell or two.
7. 7

   Clean upBench work

   Leave the microscope and the bench safe for the next person.

   The method for this part

   1. 1

      Wipe the oil off the 100× lens with lens tissue.

      Dried oil ruins the lens for the next person.

   2. 2

      Discard the slide into the disinfectant jar.

      The smear holds live organisms until it's disinfected.

   3. 3

      Turn off the Bunsen burner.

      Never leave a flame burning unattended.

What you'll take away

• The order never changes: crystal violet, iodine, decolouriser, safranin.
• Decolourising is the critical step: seconds too long turns Gram-positives pink.
• Heat-fix a dry smear, or the rinses will wash it off the slide.
• Controls prove the stain: a known Gram-positive must be violet, a known Gram-negative pink.
• Oil goes with the 100× objective only; report reaction, shape and arrangement.

Equipment unlocked

• Light microscope
• Gram stain kit
• Glass slides