Microbiology: Identify Bacteria

Bacteria is everywhere, bacteria is part of our world and ever ourselves. This is a short lesson on how to identify bacteria. You will learn staining techniques and learn how to perform biochemical tests. You will also learn to read the result and understand how to use the results to identify a bacterium.  It is important to know these skills, especially if you were to work in a diagnostics lab. Bacterial infections are all different and must be treated differently as well. If you have a Gram-negative bacteria  and mistakenly determined it was Gram-positive the treatment would not work and could kill your patient.

Aseptic Techniques

What are aseptic techniques?

Aseptic techniques are important laboratory skills in the field of microbiology. Microbiologists use aseptic techniques for a variety of procedures such as transferring cultures, inoculating media, isolation of pure cultures, and for performing microbiological tests.

Aseptic techniques are important to keep infectious microorganism from sterile surfaces or tissues. Using aseptic techniques also prevent contamination of test and or experiments in a lab.

Flaming the loop

Hold the loop in the flame of the Bunsen burner, this will kill all contaminating organisms. The sterilized loop should glow red-hot for a few seconds. After flaming, allow the loop to slightly cool t before picking up organisms from the inoculum culture.

Flaming the mouth of the test tube

Pass the mouth of a tube through the flame of a Bunsen burner to create a convection current which forces air out of the tube.  This will prevent airborne contaminants from entering the tube. The heat of the Bunsen burner also causes the air around your work area to rise, reducing the chance of airborne microorganisms contaminating your cultures. 

Agar Slants

Cultures are often transferred to agar slants, in addition to broth tubes and agar plates. An agar slant is a test tube containing agar, in which the solid agar forms a slant in the test tube.  When inoculating an agar slant, draw the loop containing the inoculum very lightly over the surface in a zigzag formation while being careful not to break the surface.

Staining Techniques

Simple Stain

Simple Stain

A simple Stain requires one dye causing the all the cell to be the same color. A simple stain is used to reveal size, shape and arrangement of cells. A simple stain takes advantage of the attraction and binding of positively charged dyes to the negatively charged surface of the cells.

Preparation of a smear and heat fixing

  1. Using a sterilized inoculating loop, transfer loopful of liquid suspension containing bacteria to a slide or transfer an isolated colony from a culture plate to a slide with a water drop.
  2. Disperse the bacteria on the loop in the drop of water on the slide and spread the drop over an area the size of a dime. It should be a thin, even smear.
  3. Allow the smear to dry thoroughly.
  4. Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times. It fixes the cell in the slide. Do not overheat the slide as it will distort the bacterial cells.

Types of simple Stain

  • Methylene blue
  • Malachite green
  • Crystal violet
  • Basic fuchsin
  • Safranin

Staining

  1. Cover the smear with one of the simple stain dyes and allow the dye to remain in the smear for approximately one minute.
  2. Using distilled water wash bottle, gently wash off the excess dye from the slide by directing a gentle stream of water over the surface of the slide.
  3. Wash off any stain that got on the bottom of the slide as well.
  4. Saturate the smear again but this time with Iodine. Iodine will set the stain
  5. Wash of any excess iodine with gently running tap water. Rinse thoroughly.
  6. Wipe the back of the slide and blot the stained surface with bibulous paper or with a paper towel.
  7. Place the stained smear on the microscope stage smear side up to observe.

What is the benefit of  a simple stain?

  • reveal size of cells
  • reveal shape of cells
  • reveal arrangement of cells
  • all of the above

Gram Stain

Gram Stain

A Gram stain is one of the most important and widely used differential staining techniques in microbiology. This technique was introduced in 1884 by Danish Physician Christian Gram.

Preparation of a smear and heat fixing

  1. Using a sterilized inoculating loop, transfer loopful of liquid suspension containing bacteria to a slide or transfer an isolated colony from a culture plate to a slide with a water drop.
  2. Disperse the bacteria on the loop in the drop of water on the slide and spread the drop over an area the size of a dime. It should be a thin, even smear.
  3. Allow the smear to dry thoroughly.
  4. Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times. It fixes the cell in the slide. Do not overheat the slide as it will distort the bacterial cells.

Dyes used in Gram stain

  • Crystal violet
  • Grams Iodine
  • Safranin

Perform Gram Stain

  1. Gently cover smear with crystal violet and let stand for 1 minute. 
  2. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. 
  3. Gently cover the smear with Gram’s iodine and let stand for 1 minute. 
  4. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. The smear will appear as a purple circle on the slide. 
  5. Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful not to over-decolorize. 
  6. Immediately rinse with water. 
  7. Gently cover with safranin to counter-stain and let stand for 45 seconds. 
  8. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. 
  9. Blot dry the slide with bibulous paper. 
  10. View the smear using a light-microscope under oil-immersion. If the bacterium appear to be red they are Gram negative bacteria, if they are appear to be purple they are gram positive bacteria.


Which of the following is a gram positive bacteria?

Which of the following is a Gram negative bacteria?

Biochemical Reactions

SIM

SIM

SIM medium is a combination differential medium that tests three different parameters, which are represented by the three letters in the name:

Sulfur Reduction

Indole Production

Motility

Inoculation

  1. Using isolated colonies from an 18-24 hour culture on solid media, inoculate the SIM Medium by stabbing the center of the medium to a depth of 1/2 inch

Incubation

  1. Incubate the inoculated medium aerobically at 35ºC. for 18-24 hours

Add Reagents

  1. Observe for H2S production and motility
  2. Once H2S and motility reaction have been read and recorded, apply three drops of Kovacs Reagent to the surface of the medium
  3. Observe for the development of a pink to red color

Interruption of the results

  • A positive H2S test is denoted by a blackening of the medium along the line of inoculation. A negative H2S test is denoted by the absence of blackening.
  • A positive motility test is indicated by a diffuse zone of growth flaring from the line of inoculation.
  • A negative motility test is indicated by growth confined to the stab line.
  • A positive test for indole is denoted when a pink to red color band is formed at the top of the medium after addition of Kovacs Reagent. A yellow color denotes a negative indole test after addition of Kovacs Reagent.

What does SIM stand for?

  • Sulfur Reduction, Indole Production, Motility
  • Sam, Is, Mad
  • Stains, Inductions, Motility
  • Saffron, Indole Production, Motility

Methyl Red and Voges-Proskauer Test

Methyl Red Test

Methyl Red (MR) test determines whether the microbe performs mixed acids fermentation when supplied glucose. Types and proportion of fermentation products produced by anaerobic fermentation of glucose is one of the key taxonomic characteristics which help to differentiate various genera of enteric bacteria.

Voges-Proskauer Test

Voges-Proskauer (VP) test determines if an organism produces acetylmethyl carbinol from glucose fermentation. 

Perform a Methyl Red Test

  1. Inoculate two tubes containing MR-VP Broth with a pure culture of the microorganisms under investigation
  2. Incubate at 35 °C for 24 hours
  3. Add about 5 drops of the methyl red indicator solution to the first tube
  4. In the second tube add Barrit's reagent for the Voges-Proskauer test 
  5. A positive reaction is indicated, if the color of the medium changes to red within a few minutes

What is the dye indicator used for the Methyl Red and Voges-Poskauer Test?

  • Voges-Poskauer
  • Methyl Red
  • Methyl Blue
  • India Ink

TSI

Triple Sugar Iron (TSI)

Triple Sugar Iron (TSI) Agar is recommended for use in the differentiation of Enterobacteriaceae by their ability to ferment glucose, lactose, and sucrose, and their ability to produce hydrogen sulfide.

Reactions in TSI should not be read after 24 hours of incubation, because aerobic oxidation of fermentation products form lactose and/or sucrose will occur and the slant will eventually revert to alkaline state.

Perform a TSI

  1. Touch a well isolated colony with a sterile straight wire
  2. Inoculate TSI by first stabbing through the centre of the medium to the bottom of the tube and then streak the surface of the slant
  3. Leave the cap loose and incubate the tube at 35 in ambient air for 18 to 24 hours
  4. Observe the reaction

What does TSI stand for?

  • Triple Sample Iodine
  • Three Sugar Inside
  • Triple Sugar Iron
  • Three Sample Iodine

Urease

Urease test

Urease broth is a differential medium that tests the ability of an organism to produce an exoenzyme, called urease. Urease hydrolyzes urea to ammonia and carbon dioxide. The broth contains two pH buffers, urea, a very small amount of nutrients for the bacteria, and the pH indicator phenol red.

Innoculation

  1. Select the Christiansen's urea broth medium  
  2. Using aseptic techniques pick up an inoculum from the culture tube
  3. Immediately transfer the inoculum into the fresh, sterile medium 

Incubation

  1. Place the inoculated tube into the 35-37 C incubator
  2. Incubate for 24 hours

Heading 1 text goes here

  1. Retrieve desired incubated culture from the incubator 
  2. Observe test result

Positive and Negative test results

  • If organism produces urease enzyme, the color of the slant changes from light orange to magenta.
  • If organism do not produce urease the agar slant and butt remain light orange (medium retains original color).

Fill in the Blank

Urease hydrolyzes to ammonia and

Catalase

Catalase test

This test demonstrate the presence of catalase, an enzyme that catalyses the release of oxygen from hydrogen peroxide (H2O2). Catalase test are used to differentiate those bacteria that produces an enzyme catalase, such as staphylococci, from non-catalase producing bacteria such as streptococci.

Slide test 

  1. Transfer a small amount of bacterial colony to a surface of clean, dry glass slide using a loop or sterile wooden stick
  2. Place a drop of 3% H2O2 on to the slide and mix
  3. A positive result is the rapid evolution of oxygen (within 5-10 sec.) as evidenced by bubbling
  4. A negative result is no bubbles or only a few scattered bubbles
  5. Dispose of your slide in the biohazard glass disposal container

Tube test

  1. Add 4 to 5 drops of 3% H2O2 to in a test tube
  2. Using a wooden applicator stick, collect a small amount of organism from a well-isolated 18- to 24-hour colony and place into the test tube
  3. Place the tube against a dark background and observe for immediate bubble formation (O2 + water = bubbles) at the end of the wooden applicator stick

Positive and negative results

  • Catalase Positive reactions: Evident by immediate effervescence (bubble formation)
  • Catalase Negative reaction: No bubble formation (no catalase enzyme to hydrolyze the hydrogen peroxide)

Which is Catalase positive?

Coagulase

Coagulate test

Coagulase is an enzyme-like protein and causes plasma to clot by converting fibrinogen to fibrin. Staphylococcus aureus produces two forms of coagulase: bound and free. Coagulase test is used to differentiate Staphylococcus aureus (positive) which produce the enzyme coagulase, from S. epidermis and S. saprophyticus (negative) which do not produce coagulase. i.e Coagulase Negative Staphylococcus.

  • Bound coagulase (clumping factor) is bound to the bacterial cell wall and reacts directly with fibrinogen. This results in an alternation of fibrinogen so that it precipitates on the staphylococcal cell, causing the cells to clump when a bacterial suspension is mixed with plasma. This doesn’t require coagulase-reacting factor.
  • Free coagulase involves the activation of plasma coagulase-reacting factor (CRP), which is a modified or derived thrombin molecule, to from a coagulase-CRP complex. This complex in turn reacts with fibrinogen to produce the fibrin clot.

Slide Test (to detect bound coagulase)

  1. Place a drop of physiological saline on each end of a slide, or on two separate slides
  2. Transfer some of the inoculum to each saline drop
  3. Add a drop of human or rabbit plasma to one of the suspensions, and mix gently
  4. Look for clumping of the organisms within 10 seconds
  5. No plasma is added to the second spot of saline, this is our control

Tube Test (to detect free coagulase)

  1. Dilute the plasma 1 in 10 in physiological saline ( mix 0.2 ml of plasma with 1.8 ml of saline)
  2. Label three test tubes: T (Test) P (Positive) and N (Negative)
  3. Pipette 0.5 ml of the diluted plasma into each tube
  4. Add 5 drops (0.1 ml) of the Test organisms to the tube labelled T, 5 drops of S. aureus culture to the tube labelled P and 5 drops of sterile broth to the tube labelled N
  5. After mixing, incubate the three tubes at 35-37 Degree Celsius
  6. Examine the clotting after one hour, if no clotting check for clotting every 30 minutes for up to six hours

Positive result

Coagulase Positive: Clot of any size

On the slide above, the spot on the left tis a positive result and on the left a negative result. 

Negative result

Coagulase Negative: No clot 

In the test tubes above, the tube on the left is a positive result and the tube on the right is a negative result

Identify the Bacterium

What does your unknown bacteria look like?

Is your unknown Gram positive or Gram Negative?

Bacteria with thick cells walls are termed gram-positive because they're susceptible to dying during a test called the Gram stain. The Gram stain is the first test used in bacterial classification. Bacteria with thin or absent cells walls are gram-negative because they do not trap the Gram stain dye.

What shape is the unknown?

  • Spherical bacteria are known as cocci, bacteria that form straight rods are called bacilli and bacteria with an intermediate shape are called coccobacilli. These can all be gram-negative or gram-positive. Rigid, spiral-shaped bacteria are known as spirilla and are only gram-negative. 
  • Cocci and bacilli are further classified by the linkages they form after cell division. Diplococci and diplobacilli stick together in pairs. Streptococci and streptobacilli form chains. Tetrad cocci stay in squares of four bacteria. Sarcinae cocci form eight-bacteria cubes and staphylococci form clusters.

Using the Biochemical Tests to Identify the Unknown

Biochemical flow charts

Once you have completed all biochemical tests and staining you can use biochemical flow charts to determine a unknown bacteria.