MICROBIAL CONTAMINATION IN HERBS AND THEIR FORMULATIONS

V.K.VIKRAM VARMA
M PHARMACY
(PHARMACOGNOSY)
SECOND SEMESTER
SPER
JAMIA HAMDARD
1

INTRODUCTION
 Microbial contamination is defined as the presence of living
microorganisms in a specified environment in general, the
microorganism will be those classified as bacteria, fungi, viruses,
 Herbal drugs normally carry a number of bacteria, & moulds often
originating in the soil. Poor methods of harvesting, cleaning,
drying, handling, & storage may also cause additional
contamination.
19-04-2020V.K.VIKRAM VARMA
2

CONTD.
 The leafy parts of plant that make up herbs will become
contaminated if they are grown in unclean water or soil. This is why
washing of herbs must be undertaken to ensure that food sold as
ready to eat items or do not harbor microorganisms.
 Contamination by microorganism can occur when fruits, roots, or
bark are dried under the sun.
3

SOURCES OF CONTAMINATION
The raw materials
including water, from
which the product is
manufactured
The manufacturing
environment including the
atmosphere, equipment
and work surfaces
Manufacturing personnel
4

SOURCES
RAW MATERIALS;
 Solid raw materials may serve as a source of contaminants which will later spoil
a formulated product
 Solids can be contaminated with E.coli and staphylococci
PACKAGING MATERIALS
 Containers of pharmaceuticals are becoming increasing elegant & made from a
large variety of materials particularly plastics. This should result in a reduction in
microbial spoilage because plastics are not biodegradable like the cellulose
materials papers & card
5

LIMITS FOR MICROBIAL CONTAMINATION
Microorganism Finished products Raw materials
E. coli 101 104
Salmonella - -
Total aerobic bacteria 105 -
Enterobacteria 103
6

LIMITS AS PER WHO
MICROORGANISM CRUDE DRUG FOR
PROCESSING
READY FOR INTERNAL
USE
READY FOR TOPICAL
USE
Salmonella _ nil nil
Enterobacteria _ 103 104
Total aerobic _ 105 107
E.coli 104 10 102
7

TYPES OF CONTAMINATION
Are of two major types;
DIRECT CONTAMINATION
 Contamination by microbial components & poorly maintained Heating,
Ventilation & Air Conditioning (HVAC) system
CROSS CONTAMINATION
 Cross contamination is how microbes can spread Passing the microbes or other
harmful substances indirectly from one sample to another through improper &
unsterelised equipments
8

DETECTION OF CONTAMINATION
TOTAL VIABLE COUNT
 The total viable count (TVC) of the herbal material being examined is
determined, using one of the following methods;
 Membrane filtration plate count or serial dilution. Aerobic bacteria & fungi
(moulds and yeasts) are determined by the TVC
Pretreatment of herbal materials
 Depending on the nature of crude herbal materials grind, dissolve, dilute,
suspend, or emulsify it using a method & eliminate any antimicrobial
properties by dilution, neutralisation or filtration
9

Some materials have special requirements which have to be met for
acceptable pretreatment to be performed some examples are as
follows
 Materials containing tannins
 Water soluble materials
 Non fatty materials
 Fatty materials
CONTD.
10

Use petri dishes 9-10 cm
in diameter to one add a
mixture of 1 ml of the pre
treated herbal material &
about 15 ml of liquefied
casein soybean digest
agar at a temperature not
exceeding 45
Spread the material on
the surface of the
liquefied medium in a
petridish dilute the
necessary material to
obtain an expected
colony count of not more
than 300.prepare atleast
two dishes using the
same dilution
Invert them & incubate
them at 30-35 for 48-72
hours count the number
of colonies formed &
calculate the results using
the plate with largest
number of colonies
TEST PROCEDURE
• PLATE COUNT (FOR BACTERIA)
11

12
• PLATE COUNT (FOR BACTERIA)
a)The pour plate method
b)The spread plate method

• FOR FUNGI
Use petri dishes 9-10 cm in diameter. To one dish add a mixture
of 1 ml of the pretreated material and about 15 ml of liquefied
glucose agar with antibiotics at a temp not exceeding 45
Spread the pretreated material on the surface of the solidified
medium in a petri dish dilute the material to obtain expected colony
count of not more than 100
Prepare atleast two dishes using the same dilution and
incubate them at 20-25 degree for 5 days, count the
number of colonies
13

SAMPLING PROCEDURE
For Plate Count Method
 Sterilize the loop & pull it. Take the culture with the help of sterile loop.
 Remove the plug near the nutrient broth containing test-tube & flame
its neck by passing it through flame
 Then, take a loop full of culture with the help of a loop and put it in test-
tube containing nutrient broth
 Replace the plug & sterilize it.
 Keep the test-tube containing nutrient broth & in an incubator for 24
hours & observe the growth
14

CALCULATIONS/RESULTS
• CFU =colony forming unit
• CFU per ml of sample=no. of colonies
counted X the dilution factor of the plate
count
15

MEMBRANE FILTRATION METHOD
 Use membrane filters with normal pore size not
greater than 0.45m & with a proven effectiveness
at retaining bacteria
 Where filters of a different diameter are used,
 Adjust the volume of the dilutions & washings than
sterilized
 Than examined under aseptic conditions, & the
membrane is then transferred to the culture
medium
16

DETAILED METHOD
Transfer 10 ml solution containing 1 gm of material to each two
membrane filter apparatus & filter immediately
Dilute the pretreated material to obtain an expected colony
count of 10-100
Wash each membrane filtering three or more sucessive
quantities of approx 10 ml of sodium chloride at ph 7.0
The surface of plate with glucose agar, incubate plates for 5
days at 30-35 for bacteria and 20-25 for detection of fungi
count the no of colonies calculate the no of microbes per gram
or per ml of material tested
18

SERIAL DILUTION
 Prepare a series of 12 tubes each containing 9-10 ml of soybean
casein digest
 First group of three tubes add 1 ml of the 1:10 dilution of dissolved
homogenized material
 Second group of the test tubes add 1 ml of 1:100 dilution of the
material
 Third group of three test tubes add 1 ml of 1:1000dilution of the
material
 Last three tubes add 1 ml of the diluent
19

 Incubate the tubes at least 30-35℃ for atleast 5 days
 No microbial growth should appear in the last three tubes
 If the reading of the results is difficult
 Prepare a subculture in a liquid or a solid medium and evaluate
the result after incubation
CONTD.
20

TEST FOR SPECIFIED MICROORGANISM
E.COLI;
 Streak a portion from enrichment culture on surface of macconkey agar
medium cover the dish & incubate
 If none of the colonies are brick red in colour sample meet the
requirment of test for absence of E.coli
PSUDOMONAS AERUGINOSA
 Inoculate 100 ml of fluid soybean casein digest medium with quantity of
solution containing 1 g or 1 ml of preparation being examined &
incubate at 35 to 37℃ for 48 hrs
 If colony forms carry out oxidase & pigment test
21

REFRENCES
 http://shodhganga.inflibnet.ac.in/bitstream/10603/117989/3/chapter%201.pdf
 https://www.ncbi.nlm.nih.gov/pubmed/26118160
 https://www.slideshare.net/DhSani1/microbial-contamination-and-detection
 https://www.slideshare.net/SaifulIslam750/microbiological-contamination-
and-preservation-of-pharmaceutical-products
 https://biologyreader.com/membrane-filtration-method.html
22

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