ISOLATION OF BACTERIA

FROM WOUND SPECIMENS

BY WAJIHA QAISER
 INTRODUCTION:
 Wounds are open skin or tissue injuries that disrupt the integrity of the body's protective
barrier and create an entry point for microorganisms.
 Common types of wounds include abrasions (scratches), lacerations (cuts), puncture
wounds (penetrating injuries) and incisions (Surgical cuts).
 Wounds can be superficial or deep, and their severity can range from mild to life
threatening.
 Superficial wounds are usually not considered serious as they are only present on the
outer layer of the skin.
 Deep wounds can be much severe.
 Wounds can become infected if the normal skin barrier is broken as it allows the
bacteria and other microorganisms to enter the body and multiply.
 Proper wound care is important to prevent the infection, promote healing and to reduce
the risk of complications.
 Treatment may include cleaning of wound, application of antibiotics or other
medications and in some cases, surgery is required to repair damaged tissues.
 IMPORTANCE OF BACTERIAL
ISOLATION FROM WOUND SPECIMENS:

 Essential process in identifying the causative agents of wound infections or Diagnosis.

 Helps in the selection of appropriate treatment.
 Essential for the monitoring of treatment response.
 Helps in determining severity of infection.
 Helps researchers in their studies and develop new treatments.
BACTERIAL SPECIES
Most commonly isolated bacterial species from wound specimens include:
 Staphylococcus aureus
 Gram-positive organism
 Causes skin infections
 Study: Arora et al.,2014 – Diabetic foot ulcers
 Pseudomonas aeruginosa:
 Gram-negative bacterium
 Causes Pneumonia, urinary tract infections and wound infections
 Study: Gardner et al., 2017- Pressure ulcers
BACTERIAL SPECIES (Cont.)
 Enterococcus faecalis:
 Gram-positive
 Part of normal gut flora that cause infection if enters other parts of the body
 Study: Eleftheriaou et al., 2015 - Chronic wounds
 Escherichia coli:
 Gram-negative
 Part of normal gut flora that cause infection if enters other parts of the body.
 Study: Johnson and Russo, 2018 – Acute pyelonephritis
 Streptococcus pyogenes:
 Gram-positive
 Causes skin and soft tissue infections
 Study: Eleftheriadou et al., 2015 – Chronic wounds
SAMPLING OF WOUND
SPECIMENS
SAMPLING
 First step in the isolation of Bacteria. Sampling of wound specimen includes several
steps:

 Wound area should be cleaned with antiseptic solution to reduce risk of

contamination.
 If the isolation is related to pathogens, appropriate protocols should be followed.
SAMPLING (Cont.)

SAMPLE COLLECTION:

 Sample of wound exudate, pus or tissue is collected

using a sterile swab.
 Rub the swab gently to ensure that it comes in
contact with any bacteria present.
 A syringe or needle is used to collect the fluid or
tissue samples from deep wounds.
SAMPLING (Cont.)

SAMPLE HANDLING:

 The collected sample is placed in a sterile container such as test tubes or petri
dishes to prevent the growth of other microorganisms.
 Wound specimens are properly labelled.
 The specimens are transported to laboratory as soon as possible while maintaining
the temperature at 4°C to prevent the overgrowth of normal flora, and for
successful isolation.
 The specimen is processed immediately to minimize the risk of contamination and
degradation of bacterial growth.
ISOLATION OF BACTERIA
FROM WOUND SPECIMENS
ISOLATION OF BACTERIAL SPECIES
 Isolation of bacterial species from wound specimens include several steps:

 COLLECTION OF WOUND SPECIMEN:

 Wound area is cleaned by an antiseptic solution.

 Sample can be collected by using swabs, tissue biopsies and aspirates.
 Sample is collected in a sterile container for transport to the laboratory.
 ISOLATION OF BACTERIAL SPECIES
 INOCULATION:
The specimen is streaked on various culture media
based on the type of bacteria. Some of the culture media
include:
 Blood agar for gram-positive cocci
 MacConkey agar for gram-negative rods
 Chocolate agar for Fastidious organisms
 Sabouraud agar for Fungi such as Candida species.
There are many other culture media used for the growth of
different bacteria based on their nutritional requirements.
ISOLATION OF BACTERIAL SPECIES

 INCUBATION:
 Plates are incubated at optimal conditions for the growth of targeted bacterial
species.
 Length of incubation period varies depending on the type of agar medium used
and the bacterial species being isolated.
IDENTIFICATION
 After 24-48 hours of incubation, the
bacteria present in sample grow and form
visible colonies on agar medium which are
then examined for their physical and
biochemical characteristics.
 Physical characteristics include color, size
and shape of colonies.
 The biochemical characteristics include the
production of enzymes and utilization of
different nutrients.
 IDENTIFICATION
 BIOCHEMICAL TESTS:
1. Gram staining
2. Catalase test
3. Methyl red (MR) test
4. Vogues-Proskauer (VP) test
5. Indole test
6. Citrate test
7. Urease test

 MOLECULAR TEST:
1. Polymerase Chain Reaction (PCR)
ISOLATION OF BACTERIA
FROM WOUND SPECIMENS
Enterococcus faecalis
ISOLATION OF Enterococcus faecalis

 Gram-positive
 Commonly present in GIT of humans and animals
 Generally considered harmless but cause infection in people with weak immune
system.

 SAMPLE COLLECTION:
 Sample is collected using sterile swab or biopsy(a small piece of tissue from the
wound is collected using a sterile scalpel).
 Sample transferred to a sterile container for transport to lab.
ISOLATION OF Enterococcus faecalis:

 INOCULATION:
 The specimen is plated on a suitable culture medium.
 Bile esculin agar and blood agar are commonly used.

 INCUBATION:
 Culture plates are incubated at 35-37°C for 24-48 hours.
ISOLATION OF Enterococcus faecalis:

 IDENTIFICATION
 Small, grayish white and translucent colonies on blood agar.
 Black, shiny colonies on Bile esculin agar.

 Further identification is done by

 Gram staining- Purple cocci.
 Catalase test-Catalase negative.
 Bile esculin test- Produce black color
 Antibiotic susceptibility test- Susceptible to Vancomycin,
Streptomycin and Ampicillin.
ISOLATION OF BACTERIA
FROM WOUND SPECIMENS
Klebsiella pneumoniae
ISOLATION OF Klebsiella pneumoniae:

 Gram-negative
 Family Enterobacteriaceae
 Commonly present in human digestive system, skin and feces.

 SAMPLE COLLECTION:
 Sample collected by using sterile swab or syringe and needle.
 Swab rubbed over the wound surface to collect sample.
 Syringe is used to aspirate the wound fluid.
 Samples are placed in sterile container and transported to laboratory.
ISOLATION OF Klebsiella pneumoniae:

 INOCULATION:
 The specimen is plated on a suitable culture medium.
 MacConkey agar, Eosin Methylene Blue(EMB) agar and blood agar are
commonly used.

 INCUBATION:
 Culture plates are incubated at 37°C under aerobic or microaerophilic conditions.
ISOLATION OF Klebsiella pneumoniae:

 IDENTIFICATION:
 Colonies appear pale-white to light pink. Some
strains may appear more yellowish and
pigmented on Blood agar.
 Produces metallic green colonies on EMB agar.
 Produces pink colonies on MacConkey agar.
 ISOLATION OF Klebsiella pneumoniae:

 Further identification is done by;

 Gram-staining- Pink, short rods
 Indole test- Indole negative
 Methyl-red test- Methyl red positive
 Vogues-Proskauer test- VP negative
 Citrate test- Citrate positive
 Urea hydrolysis test- Positive for urea hydrolysis test
 Polymerase Chain Reaction (PCR)
ISOLATION OF BACTERIA
FROM WOUND SPECIMENS
Streptococcus pyogenes
ISOLATION OF Streptococcus pyogenes:

 Gram-positive
 Family Group A Streptococcus (GAS)
 Causes Impetigo, pharyngitis and Toxic Shock Syndrome.

 SAMPLE COLLECTION:
 A throat or skin lesion sample from patient is collected through sterile swab.
 Samples are placed in sterile container and transported to laboratory for
processing.
ISOLATION OF Streptococcus pyogenes:

 INOCULATION:
 The collected sample is inoculated onto an appropriate culture medium.
 Blood agar supplemented with 5% sheep blood is the most commonly used
medium.

 INCUBATION:
 The culture plates are incubated at 37°C under aerobic conditions with 5-10% CO₂
for 24-48 hours.
 ISOLATION OF Streptococcus pyogenes:

 IDENTIFICATION:
 Streptococcus pyogenes produce grayish-white, translucent
colonies with a narrow zone of beta-hemolysis around them on
blood agar.
 Further confirmation is done using:
 Gram staining- Purple cocci.
 Catalase test- Catalase negative
 Bacitracin susceptibility test- Sensitive to bacitracin, forms zone
of inhibition.
 Rapid Antigen Detection Test(RADT)
 PCR
FACTORS AFFECTING RESULTS OF
BACTERIAL ISOLATION:

 Sampling technique.
 Transportation of sample.
 Choice of culture media.
 Inoculation technique.
 Incubation conditions.
 Interpretation of results.
PRECAUTIONS:

 Proper sample collection.

 Use of appropriate PPE.
 Proper handling and disposal of sample.
 Use of appropriate disinfectants.
 Proper sterilization of instruments.
 Use of appropriate agar media.
 Proper incubation should be provided.
CONCLUSION

 In conclusion, the isolation of bacteria from wounds is an important step in identifying

the causative agents of infections. Staphylococcus aureus, Pseudomonas aeruginosa,
Enterococcus faecalis, Escherichia coli, and Streptococcus pyogenes are some of the
bacterial species commonly associated with wound infections. Proper identification
and characterization of these bacteria can help guide appropriate treatment and
improve patient outcomes.
THANK YOU!