Pus Having Streptococcus pyogenes: Introduction and its Gram Stain
Streptococcus pyogenes in Pus
Pus having Streptococcus pyogenes as shown above image. Pus is an exudate, typically white-yellow, yellow, or yellow-brown, formed at the site of inflammation during infection (bacterial or fungal ). An abscess is an accumulation of pus in an enclosed tissue space whereas a visible collection of pus within or beneath the epidermis is a pustule, pimple, or spot. Pus consists of a thin, protein-rich fluid and dead leukocytes from the body’s immune response (mostly neutrophils).
Pyogenic ( pus-forming) Bacteria
Following common bacteria are responsible for the production of pus and they are-
#How to test pus culture and sensitivity a shown below-
Gram Stain of Pus
Principle of Gram Stain
The reaction is dependent on the permeability of the bacterial cell wall and cytoplasmic membrane, to the dye–iodine complex. In Gram-positive bacteria, the crystal violet dye iodine complex combines to form a larger molecule which precipitates within the cell. The alcohol /acetone mixture which acts as a decolorizing agent causes dehydration of the multi-layered peptidoglycan of the cell wall. This causes a decrease in the space between the molecules causing the cell wall to trap the crystal violet iodine complex within the cell. Hence the Gram-positive bacteria do not get decolorized and retain primary dye appearing violet.
Also, Gram-positive bacteria have more acidic protoplasm and hence bind to the basic dye more firmly. In the case of Gram-negative bacteria, the alcohol, being a lipid solvent, dissolves the outer lipopolysaccharide membrane of the cell wall and also damages the cytoplasmic membrane to which the peptidoglycan attaches. As a result, the dye-iodine complex does not retain within the cell and permeates out of it during the process of decolonization. Hence, when a counterstain uses, they take up the color of the stain and appear pink.
Requirements for Gram Stain
a) Compound light microscope
b) Reagents and glasswares
Clean grease-free slides
Crystal violet (Basic dye)
95% ethanol (decolorizing agent)
1% safranin or dilute carbol fuchsin or neutral red
Take a clean, and grease-free slide for making a smear.
Take a loopful of 0.85% saline i. e. physiological saline and place it on the Center of the slide.
With a loop take pus and emulsify in the saline drop forming a thin film.
Allow the smear to air dry.
Heat fix the smear while holding the slide at one end, and by quickly passing the smear over the flame of the Bunsen burner two to three times.
Procedure of Gram Stain
Cover the smear with crystal violet and allow it to stand for one minute.
Rinse the smear gently under tap water.
Cover the smear with Gram’s iodine and allow it to stand for one minute.
Rinse smear again gently under tap water.
Decolorize the smear with 95% alcohol.
Rinse the smear again gently under tap water.
Cover the smear again gently with safranin for one minute.
Rinse the smear again gently under tap water and air dry it.
Observe the smear first under the low power (10X) objective, and then under the oil immersion (100X) objective.
Observation of Gram Stain
Positive Control: violet color, round in shape in single, pairs and cluster
Test: red color and rod in shape
Negative Control: red in color and rod in shape
Result and Interpretation of Gram Stain
Gram-positive: purple or violet color
Gram-negative: Pink or red in color
Cocci: round in shape
Bacilli: rod in shape
Positive Control(PC): Gram-positive cocci in single, pairs and cluster
Test: Gram-negative bacilli
Negative Control(NC):Gram-negative bacilli as shown above image.
E. coli under microscope|| Gram stain ||Gram Negative bacilli or Gram-negative rods as shown below-
Variety of bacteria under the microscope showing Gram-positive bacteria, gram-positive cocci in singles, pairs, clusters i.e. Staphylococcus aureus Gram-positive rods or bacilli Bacillus species Gram-negative bacilli or rods Salmonella Typhi Diphtheroids Sputum gram stained smear having ideal smear Gram-positive cocci in pairs inside the pus cell Gram-positive cocci in chains
A patient was 53 years old with Chronic Otitis Media (COM) having a failure of antibacterial drugs- Pus swab from ear discharge sent to microbiology section for Gram staining- result found – Fungal spores with plenty of pus cells and lacking bacteria as shown in the video.
Strongyloides stercoralis under Gram stained slide of sputum- a very very rare case-
Impression for this a rare case report from Gram stain of sputum While reporting gram stained slide, we always remember the nature of specimens and availability of organisms; and broaden our vision for report variety of causative agents without missing them because as you know laboratory diagnosis is the third eye of clinicians that makes treatment easier as well as short hospital stay and reduce patient economic burden.
Growth of Candida albicans on SDA and its Gram-stained smear under the microscope and germ tube test (GTT) positive as shown below-
Staphylococcus aureus is the most common cause of boils.
Some disease processes caused by pyogenic infections are impetigo, osteomyelitis, septic arthritis, and necrotizing fasciitis.
Pus is a sterile specimen.
Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
Clinical Microbiology Procedure Handbook Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.