Sterility testing, Pyrogens, and Pyrogen

testing

Kinds of Media

MEDIA EXAMPLES
1. BASAL MEDIA Examples: Nutrient broth,
nutrient agar and peptone water.
Staphyloco ccus and Enterobacteriaceae
grow in these media.

2. ENRICHED MEDIA Enriched media are blood agar and

Lowenstein-Jensen media. Streptococci
grow in blood agar media.

3. SELECTIVE MEDIA Examples: MacConkey agar,

Lowenstein-Jensen media, tellurite
media (Tellurite inhibits the growth of
most of the throat organisms except
diphtheria bacilli). Antibiotic may be
added to a medium for inhibition.

4. INDICATOR (DIFFERENTIAL) Examples: Blood agar and

MEDIA
5. TRANSPORT MEDIA
6. STORAGE MEDIA
MacConkey agar are indicator media.
Examples: Cary-Blair medium, Amies
medium, Stuart medium.
Examples: Egg saline medium, chalk
cooked meat broth.

Bacterial Growth and Growth media

Nutrient Agar.
 It is solid at 37°C. 2.5% agar is added in nutrient broth.
 It is heated at 100°C to melt the agar and then cooled.

Blood Agar.
 Most commonly used medium.
 5-10% defibrinated sheep or horse blood is added to melted agar at 45-50°C.
 Blood acts as an enrichment material and also as an indicator.
  Types of changes : (a) beta (p) haemolysis. The colony is surrounded by a
clear zone of complete haemolysis,

Peptone Water.
 Peptone 1% and sodium chloride 0.5%.
 It is used as base for sugar media and to test indole formation.

Chocolate Agar or Heated Blood agar.

 Prepared by heating blood agar.
 It is used for culture of pneumococcus, gonococcus, meningococcus and
Haemophilus.
 Heating the blood inactivates inhibitor of growths.

MacConkey Agar.
 Most commonly used for enterobacteriaceae.
 It contains agar, peptone, sodium chloride, bile salt, lactose and neutral red. It
is a selective and indicator medium

(1) Selective as bile salt does not inhibit the growth of enterobactericeae but
inhibits growth of many other bacteria.

(2) Indicator medium as the colonies of bacteria that ferment lactose take a
pink colour due to production of acid. Acid turns the indicator neutral red to
pink. These bacteria are called 'lactose fermenter

What is Sterility Testing?

Sterility testing attempts to reveal the presence or absence of viable micro-

organisms in a sample number of containers taken from batch of product. Based on
results obtained from testing the sample a decision is made as to the sterility of the
batch.

 Immersion (Direct Inoculation)

The immersion (direct inoculation) method requires the test article be

inoculated directly into test media.

 Membrane Filtration

The membrane filtration method requires the test article to first pass through a
size exclusion membrane capable of retaining microorganisms. The filter is
rinsed and then the membrane is transferred into the appropriate test medium
 Sampling

minimum, in accordance with the pharmacopoeial method followed
 should be selected from at least the potentially coolest part of the load.

Media Types

 Soya-bean casein digest (SCD)

 fluid thioglycollate media (FTM)

Incubation period
 atleast 14 days
 If the product produces a suspension, flocculation or deposit in the
media, suitable portions (e.g. 2-5 percent) of the contents of the containers
should be transferred to fresh media under clean room conditions, after 14
days, and reincubated for a further 7 days.

Complications that may arise once a not sterile parenteral product is

administered into your bloodstream:

• impairment of microcirculation
• blockages of blood vessels
• damage to various organs
• Phlebitis

Pyrogens
 are fever producing substances, which are metabolic products of
microorganisms which maybe present from water, solute and
equipment or introduced from manufaturing process.
 Thermostable endotoxins from cell walls of gram negative organisms

Pyrogen Test
 Also known as rabbit test
 It involves measuring the rise in temperature of rabbits following
administration of a test solution

Notes:
 Test animals
 Uses three healthy mature rabbits whose body temperature does not vary by
more than 1C from each other
 Do not use any rabbit having a temperature exceeding 39.8
 Procedure:

1. Get the baseline temperature of the rabbits

2. Inject into an ear vein 10 mL/kg of the test solution, in portions, completing each
injection within 10 minutes after start of administration
3. Record the temperature after 1 hour, 2 hours, and 3 hours
4. Observe the rise in temperature

Interpretation:

ACCEPT IF:

 No rabbit shows an individual rise in temperature that is ≥0.6 C (0.5C)

 Sum of the three individual temperature should not exceed 1.4C
 Otherwise, repeat test using additional 5 rabbits

REJECT IF:
× More than 3 out of 8 rabbits showed a temperature rise of ≥0.6C(0.5C)
× Sum of the readings of temperature rise for the 8 rabbits is more than 3.3 C

Complications that may arise once a pyrogen in a parenteral product is

administered into your bloodstream:

• chills, body aches, a rise in blood pressure, and possibly a state of shock and
death.
• Examples of these changes are shown by a reduction followed by an increase in
the number of white cells, tumor hemorrhages, and changes in venous
pressures.

ADVANTAGES:
1. It can be replicated and demonstrate the production of fever in humans
2. Detects all kinds of injectable Pyrogen unlike LAL test

DISADVANTAGES:
1. Time consuming
2. Expensive procedure
3. It is pass/fail test than assay
4. It cannot be used to test certain drugs that depresses the fever
5. Tolerance of certain class of drugs can develop in rabbits & also biological
variations are observed

Bacterial Endotoxin LAL test

 Limulus Amoebocyte Lysate test

  A test for estimating the concentration of bacterial endotoxins that maybe
present in a sample
 It causes LAL obtained from aqueous extracts of the circulating
amoebocytes of the horse shoe crab (Limulus polyphemus) which has
been prepared and characterized as a LAL reagent

Horseshoe crab
- are extremely important to the biomedical industry because their unique, copper-
based blue blood contains a substance called Limulus amebocyte lysate. The
substance, which coagulates in the presence of small amounts of
bacterial toxins, is used to test for sterility of medical equipment and virtually all
intravenous drugs.

Aim of LAL test

- is to measure quantitatively the amount of bacterial endotoxins in a given sample
of parenteral.

Methods of LAL test

 Turbidimetric LAL test

Measures the presence of endotoxins

 Colorimetric LAL test

Development of color indicates the presence of endotoxins

 Gel Clot technique

Based on gel formation

** Sources have been water which somehow entered into the manufacturing process
may produce endotoxins

Interpretation of results:

The given sample complies with endotoxin test if the positive product control gives
positive result and the negative as well as the test solution gives negative result.

The test is not valid if the product positive control is negative or the negative control is
positive or both of these conditions occur.

Retest: If the positive test is found for one of the test in the duplicate, the test is
repeated as above
PROCEDURE

• The most common approach to endotoxin testing is the limulous amoebocyte

lysate test (LAL test). This assay is based in the biology of the horseshoe crab
(Limulous). These animals produce LAL enzymes in blood cells (amoebocytes) to
bind and inactivate endotoxin from invading bacteria.

• LAL serves as a primitive immune system. Inactivation of endotoxin also forms a

clot, which can further protect the horseshoe crab from infection. The LAL test
exploits the action of this enzyme, by adding LAL reagent to the tested product,
and assaying for clot formation.

NOTE

• For parenteral products, inspections have shown that where pyrogen problems
were found in dosage forms, and when the source was one of the raw materials,
it was the active drug substance. This was particularly true for drug substances in
which process water was used at some late stage in the synthesis process. .

• As with parenteral drug products, sterile devices have occasionally been shown
to be contaminated with endotoxins. Sources have been water which somehow
entered into the manufacturing process.

ADVANTAGES

1. In vitro test
2. More sensitive
3. Easier to perform
4. Can give quantitative results
5. Less time consuming
6. Less expensive

LIMITATION

1. Specific for gram negative pyrogens only

2. Clotting enzyme is heat labile, pH sensitive
3. Possible interference problems