Staining Procedures For Identification

Organisms can be tentatively identified by growing them on media and looking at morphology or growth characteristics.  This gives the researcher a tentative idea as to what the bacteria might be but further test need to need be done to positively identify organism and many times bacterial cultures can look similar to yeast.  The next step to identifying an organism is staining and there are a variety of stain used in Microbiology, many time based on morphology, all with the purpose of helping to identify an organism.  Some stains, like wet mounts and the simple stains, give limited information while others (Gram Stain, Acid Fast Stain, Spore Stain) give the user more detailed information.

Unknown Yeast contaminant on TSA agar after 3 weeks refrigerated temperatures. The colony appeared mucoid colony with lobate edges, was white in the center of the colony and yellow on the edges.   The agar surrounding the colony had what appeared to be a brown coloration, possible diffusible pigment. Without further testing this could be mistaken for a bacterial culture.  A wet mount using Lactophenol cotton blue was performed and round cells in groups of 2-5 cells were seen with budding.  This type of staining does not stain the cell but the background with the cells appearing clear.  This is good for cell morphology, size  and arrangement.

Unknown Yeast contaminant seen on Casein media. The contaminant was around the edges of the plate with isolated colonies seen towards the center of the plate after 1 month refrigerated temperatures. Colonies were light opaque pink, mucoid and had concentric rings slightly elevated in the center. A gram stain was done to confirm yeast. Many times it can be difficult to tell if the colony you are working on is bacterial or fungal (yeast) so a Gram stain is performed.  In this case the Gram stain only gives you the basic information of cell arrangement and size since this is a yeast.  As seen by the microscope images the cells are larger then bacterial cells, ovoid and budding is observed.

Red yeast from environmental sample grown at room temperature, probably Rhodotorula spp. Gram stain showed budding, mickey mouse style, cells.

(A)

(A)  A simple stain done on a stock culture of Aquaspirillum serpens to look at cell shape. This culture was grown in TSB at room temperature, ~21 degrees C, for several     months. Aquaspirillum is a slow grower normally found in pond water. A characteristic spiral rod can be seen in the center of the slide. Aquaspirillum can be found in pond water samples and is usually not a health issue.

(B)  Simple stain done on an unknown bacteria, showing feathery rhizoid growth, Bacillus spp, on TSA after a 48 hr incubation at 37 degree’s C, isolated from a floor swab. Single bacillus and diplobacillus can be seen though out.

Gram Stain

A series of stains are used in the gram stain to distinguish between the chemical differences in bacterial cells walls.  The gram stain consist of : crystal violet (primary stain), iodine (mordant, causing a chemical complex to form between iodine and crystal violet), ethanol (decolorizer, washes away the crystal violet/iodine complex form G(-) cells, safranin (secondary stain, stains G(-) cells red after decolorization.

An organism can be classified as Gram (+) or Gram (-) base on their cell wall characteristics.  Gram (+) organisms have a thick peptidoglycan layer which holds the crystal violet /iodine complex durning the decolorization step, cells remain purple.  Gram (-) cell have a thin peptidoglycan layer and can not retain the crystal violet/Iodine complex during decolorization.  Gram (-) cells are clear after the decolorization step and are counter stained with the secondary stain, safranin, turning the cells red/pink.  Knowing cell wall structure can be useful in antibiotic treatment.

SSStreak plate isolation of a mixed culture of Staphylococcus aureus, coagulase (-) ( white circular colonies) and Serratia marcesens (red colonies).  Gram stain of S. aureus seen below.  S. aureus is seen as grape like clusters ofGram (+) coccus

 S. aureus is seen as grape like clusters of Gram (+) coccus

St

Streak plate of Micrococcus luteus (yellow circular colonies) and E. coli (white/opaque colonies)  Gram stain was done on E. coli, see image below.

E. coli: Gram (-) rod shaped bacteria that can be found in the lower intestine of animals and can be seen as a indicator organism of fecal contaminate in water samples. 

   

Steak plate isolation of Streptococcus pyogenes seen on blood agar with beta hemolysis.  S. pyogenes and can cause throat and skin infections. Gram stain on S. pyogenes see image below am stain of Streptococcus pyogenes.  The ovoid shaped cells form long distinctive chains ("strepto" greek for twisted chain), see image below. 

Gram stain of Streptococcus pyogenes.

Streak plate isolation of Bacillus subtilus.  This organism spreads fast and can have a dull dry look with lobate or irregular edges.

Gram stain of Bacillus subtilis.  Gram (+) streptobacillus, chains of rod shaped bacteria. 

Organism seen on a swab taken from a door key.  Mounding, mucoid circular colony.

Gram stain of above colony showing G+ coccus with a tetrad arrangement, coccus in a group of 4.  This is more then likely Micrococcus or Sarcinia spp both common in the environment. 

Knowing the cell wall structure can help with antibiotic choice.  Some antibiotics work better on Gram (-) and other work better on Gram (+) organisms as can be seen by the Kirby-Bauer test below.  The Kirby -Bauer test is a standardized test that measures an organisms susceptibility to different antibiotics.  Clear zones result around a disk if an organism is sensitive to that particular antibiotic.   Different antibiotic affect cells differently from inhibition cell wall synthesis to disruption of DNA/RNA sysnthesis.  By looking at the two plates below you can get an idea of which anitibiotic work better on G(+) and G (-) organisms.

Kirby-Bauer test performed on S. aureus, coagulase (-).  Most of the antibiotics except for sulfur have some effect on the S. aureus, Gram (+).

Kirby Bauer test performed on E. coli.  This organism is Gram (-) and is more resistant to antibiotic then S. aureus.

Measuring A Zone of Inhibition for the Kirby-Bauer Test

• Radius:  Measure half the distance of the zone, ie measure from the edge of the clear zone to the middle of the antibiotic disc, then multiply by 2.  Use this method  when part of the zone is not clear or has grown into another zone.

• Diameter:  Measure the entire length of the zone, from one edge of the clear zone directly across to the other edge of the clear zone.  
By measuring the zone of inhibition and using manufacture charts you can determine the level of an organisms sensitivity to the antibiotic.  Generally the larger the zone the more effective the antibiotic.

Spore Stain

  Spores, a structure produced by some bacterial species to resist harsh environmental conditions, are produced by some bacteria (Bacillus and Clostridium) as a result of unfavorable growth conditions.  Spore stain using malachite green on Bacillus cereus.  Since the tough spores produced by Bacillus spp do not stain with the gram stain we use a water bath and steam the malachite green to penetrate the hard outer surface of the spore.  Safranin is then used as a counter stain to stain the vegetative rod shaped Bacillus cells.  In the above image greenish blue endospores are seen within the vegetative cells as well as outside of the vegetative cells (released spores). Since the tough spores produced by Bacillus spp do not stain with the gram stain we use a water bath and steam the malachite green to penetrate the hard outer surface of the spore.  Safranin is then used as a counter stain to stain the vegetative rod shaped Bacillus cells.  In the above image greenish blue endospores are seen within the vegetative cells as well as outside of the vegetative cells (released spores).

Acid Fast

Mycobacterium smegmatis grown on TSA for 72 hrs at 37 degree's C.   Colonies have a distinctive wrinkled growth and take an extended time for good growth due to the production of mycolic acids which makes the colonies waxy and hard to stain.  If a gram stain is done then the Mycobacterium smegmatis cells usually do not stain.  If they did then the bacillus would be Gram (+).  Since the cells are so difficult to stain we use the acid fast stain using carboly fuschin and steam to penetrate the waxy cells, see image below.

Acid Fast stain done on a mix of Staphylococcus aureus and Mycobacterium smegmatis. The stain was done on a culture of M. smegmatis that was incubated on a TSA slant with 1ml TSB added at 37 degree's C for 4 days, S. aureus was incubated in TSB at 37 degree's C for 24 hrs. Several loops of each culture was placed on the slide and mixed. The slide was heat dried for 15 min on a hot plate set on low. Carbol fuchsin was applied to the smear and set over a steaming water bath for 10 min to help penetrate the mycolic acid in the cell wall (mycolic acid is a highly waxy substance produced by the bacteria for protection that makes gram staining difficult.  M. smegmatis would be considered a G (+) organism if an accurate gram stain were obtained), then rinsed with acid alcohol which washes the carbol fuchsin out of all but the acid fast cells. Methylene blue was then used, simple stain, as the counter stain. M. smegmatis (acid fast) stained bright pink and S. aureus blue.

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