Bacterial Identification

Gram`s Stain
Commonly used to examine the material from

Sterile body fluids [CSF, Pleural fluid & urine]

Abscesses, Wounds, Sputum and Tissues

Does not allow exact identification of organism

Determines shape and staining characteristics of

organisms
Bacterial Identification
Gram`s Staining Technique

A slide is dried with sample (fixation)

Stained with crystal violet and then iodine
Slide is decolourised with acetone/alcohol
Counter stained with Safranin (a pink dye)
Bacterial Identification

Purple - Gram Positive

Pink - Gram Negative
 Cocci [round shaped)
Positive cocci Negative cocci
Staphylococcus species
S. Aureus Moraxella catarrhalis
S. Epidermidis Nisseria meningitidis
Nisseria gonorrhoeae
Streptococcus species
S. Pneumoniae
S. Pyogens

Enterococcus Sp
E. faecalis
 Bacilli [rod shaped]
Positive bacilli

Bacillus anthracis
Listeria monocytogenes
Corynebacterium diphtheriae
Clostridium Sp.
 Bacilli [rod shaped]
Negative bacilli

Enteric Non Enteric

Bacteroides fragilis Acinetobacter Sp.
Camphylobacter jejuni
Haemophilus influenzae
Enterobacter aerogenes
Legionella pneumophila
E. coli
Klebsiella pneumoniae P. Aeruginosa
Salmonella Sp. Xanthomonas maltophilia
Shigella Sp.
Proteus mirabilis
 Culture and Identification
Bacteria are identified by

Gram Stain
Growth characteristics (type of media, aerobic & anaerobic)
Colonial morphology
Colour
Biochemical profile
 Culture and Identification
lthough many media can be used, the type of media chosen should be
based on the usual microorganism isolated

Some common agars,

Blood agar - Haemophilus, N. Gonorrhoeae
Enteric agar (e.g., MacConkey) - Gram negative
bacilli but inhibit gram positive cocci
Other enteric agar – aerobic (Gram positive
bacteria, salmonella and shigella), anaerobic
( Bacteroides fragilis)
 Common findings of infection

Clinical
• Localized
• Systemic

Laboratory
 Clinical findings of infection

Localized

Inflammation at site of infection

Swelling and warmth
Redness & Pain
Sputum production and cough
Diarrhea
Vaginal and Urethral discharge
Skin lesions
 Clinical findings of infection
Systemic

Fever, Chills and Rigors

Increased heart rate
Increased Respiratory rate
Malaise
Mental status changes
Hypotension
Laboratory findings of infection

Laboratory

Increased WBC count

Positive cultures and / or Gram stain
Increased ESR
Positive antigen or antibody titers
Antimicrobial Susceptibility Testing
Before review of the methodology and interpretation of these data , two
terms must be defined

Minimal inhibitory concentration (MIC)

MIC is the lowest antibiotic concentration that inhibits visible growth of bacteria

Minimal bactericidal concentration (MBC)

MBC is the lowest antibiotic concentration that kills 99.9% of the bacteria
Antimicrobial Susceptibility Testing
Methods commonly used for susceptibility testing of organisms

Aerobic
1. Broth dilution (Automated microdilution) method
2. Disk diffusion method

Anaerobic
1. Agar dilution
2. Broth microdilution
3. Determination of beta lactamase production
 Aerobic Bacterial Tests

Broth dilution
H. influenzae
N. gonorrhoeae
S. pneumoniae
 Aerobic Bacterial Tests

Advantage:

Ease of use
Accuracy
Reproducibility
Not expensive
 Aerobic Bacterial Tests
Disc diffusion test

The most widely used method

Paper discs impregnated with standard quantity of

antimicrobial agent are applied to the surface of an
agar plate that has been inoculated with the
suspension of the organism to be tested

The antimicrobial agent diffuses from the paper disc

through the agar in a continuously decreasing
gradient
 Aerobic Bacterial Tests
After 16-20 hours of incubation, a concentric zone of
growth of inhibition around the paper can be
measured

The large zones of inhibition are associated with

susceptibility of the organism to the antibiotic

Small or absent zones with resistance

 Aerobic Bacterial Tests

Clinical application:

- Enterobacteriaceae, Staphylococcus, Pseudomonas

- Sterile body fluids such as blood and C.S.F.

 Aerobic Bacterial Tests

Limitations:

Anaerobes cannot be tested

Fungal susceptibility

Standards for interpreting zones of growth

inhibition are based on achievable serum level of
antimicrobials
 Aerobic Bacterial Tests
Limitations:

Disc diffusion is not always applicable to

urinary tract isolates, because the achievable
levels of certain antibiotics in urine are much
higher than the serum

Bactericidal activity cannot be tested because

the disc method yields bacteriostatic only
 Anaerobic Bacterial Tests

Susceptibility testing of specific anaerobic isolates

can assist in the management of patients with
selected infections such as serious anaerobic
infections including endocarditis, brain abscess,
bone and joint infections
 Anaerobic Bacterial Tests

Methods:

1. Agar dilution
2. Broth microdilution
3. Determination of beta lactamase
production
 Anaerobic Bacterial Tests
Beta- lactamase test: [Acidometric method]
A positive beta-lactamase test predicts resistance to
Penicillin, Ampicillin, and Amoxycillin among Haemophillis
species, N. gonorrhea and Moraxella catarrhalis

For Staphylococci and Enterococci a positive beta-lactamase

tests predicts resistance to Penicillin, Ampicillin, Ticarcillin
and Pipercillin

Many bacteria produce beta-lactamase enzymes that can

inactivate numerous beta-lactum containing antibiotics
 Interpretation
Sensitive [Susceptible]:
Infection due to the tested strain will respond to an appropriate
dose of antibiotic

Resistant:
Indicates that strain is not completely inhibited by
antimicrobial concentrations within the therapeutic range

Intermediate:
Indicates that a clinical response may occur if unusually high
concentration of relatively non toxic antibiotics can be achieved
at the site of infection
Empiric Antimicrobial Therapy
Factors to consider:

Known or most probable sites of infection

The organisms that are most likely causing

infection at that site.

The usual antimicrobial susceptibility patterns of

the suspected infecting bacteria
 Empiric Antimicrobial Therapy

Gram positive cocci

S. aureus - Skin, Soft tissue, IV catheter Prosthetic
device
S. Pneumonia - Pneumonia

Gram negative cocci

N. Meningitidis – Bacteremia and meningitis
N. Gonorrhoeae - Urogenital infections
 Empiric Antimicrobial Therapy
Gram positive bacilli

L. Monocytogenes - meningitis and bacteremia

C. Difficile – Diarrhea and colitis
C. Perfringens – intra abdominal infections & bacteremia
 Empiric Antimicrobial Therapy
Gram negative bacilli

K. Pneumonia - UTI, RTI, intra abdominal

Salmonella - Diarrhea
E. Coli - UTI, bacteremia, intra abdominal infections
B. Fragilis - intra abdominal infections
E. Aerogenes - intra abdominal infections and bacteremia
P. Mirabilis - UTI, bacteremia, intra abdominal
infections
Monitoring Antimicrobial Therapy

Two general types of in-vitro tests are used to monitor antimicrobial

therapy.

1. Measurement of blood or body fluid antibiotic

activity against the responsible organism

2. Assay of actual antibiotic concentrations in blood

or other bodily fluids
Monitoring Antimicrobial Therapy
1. Serum bacterial test (SBT):

Determines the “Killing power" of patient serum

against the infecting organisms

Serum is usually obtained from the patient to

correlate with the maximum or minimum
antibacterial activity. Patients' serums are
inoculated with a standard quantity of the infecting
organism
Monitoring Antimicrobial Therapy

Clinical Applications:

The SBT has been used to monitor antimicrobial

therapy in patients with serious systemic infections
like endocarditis, osteomyelitis, and bacterimia
Monitoring Antimicrobial Therapy

2. Antimicrobial levels:
May be obtained to assess the adequacy of the chosen dose and route of
administration and to avoid toxicity.

(i) Bioassays
(ii) Immunological assays
(iii) HPLC
Monitoring Antimicrobial Therapy
Clinical Applications:

To monitor therapy with an antibiotic that could

have toxic side effects particularly in the presence of
altered hepatic or renal function

When an infection due to a sensitive organism is not

responding to antibiotic and all other therapeutic
approaches have been optimized
Thank you