I. OBJECTIVES
1
•To demonstrate good aseptic technique in culture transfer or inoculation and in
handling sterile materials.
•To demonstrate skil in isolation of organisms from a mixed culture using selectve
and differential media.
•To isoate microorganisms from a wide variety of sources and describe their colonial
morphology.
II. INTRODUCTION
Most environments carry a mixed microbial population. To fully appreciate the
contribution of each group of organisms to the ecology of the mass, one must first
dissect this mixed culture to obtain single colonies. The single colony is transferred
(picked) to a fresh medium to obtain a larger, homogeneous culture that may be studied
and characterized by a variety of techniques. One suchtechnique is caled aseptic
technique. Microbiologists and health workers use this technique to prevent
contamination of cultures from outside sources and to prevent the introduction of
potential disease agents into the human body (nfection can occur through contamination
of your hands and clothing with material from your bacterial cultures).
Aseptic Techniques
Aseptic techniques (also called sterile techniques) are defined as the processes
required for transferring a culture from one vessel to anotherwithout introducing any
additional organisms to the cuture or contaminating the environment with the culture.
The following conditions must exist for aseptic technique to be successful:
1. The work area must be wiped with an antiseptic to reduce the number of potential
contaminants.
2. The transfer instruments must be sterile.
3. The work must be accomplished quicky and efficiently to minimize the time of
exposure during which contamnation of the culture or laboratory worker can occur.
Developing a thorough understanding and knowledge of aseptic techniques and
culture transfer procedures is a prerequisite to working with microbiological cutures. You
wll save yourself a lot of time and energy and avoid erroneous results if a few simple
and common sense rules are observed when working with cultures.
Dr. Eby Bassiri ebassiri@sas.upenn.edu
Booking open Available Pune Call Girls Ambegaon Khurd 6297143586 Call Hot In...
HCM 124 lecture 9 medical entomology.pptx
1. HCM 124: MEDICAL PARASITOLOGY
AND ENTOMOLOGY
JACKSON C. KORIR (PhD)
0721- 413 606
jcheruiyot@mmust.ac.ke
Masinde Muliro University
of Science and Technology
(MMUST)
University of Choice
9. 9
Modes of transmission
1. Direct contact:
Arthropods are directly transmitted
from man to man through close
physical contact
2. Mechanical transmission:
The disease agent is just mechanically
carried by the Arthropod
10. 10
Contd…
3. Biological transmission:
Propagative: The disease agent
multiplies in number in the body of
arthropod without under going
developmental change
Cyclopropagative: The disease agent
undergoes both developmental change
and multiplies in number
Ex. Malaria parasite in Anopheles
11. 11
Cyclo developmental: The disease agent
undergoes only developmental change (no
multiplication)
Ex. Filaria larva in culex and Guinea worm
embryo in cyclops
12. 12
Cont..
Trans ovarian transmission:
The disease agent is passed onto the next
generation through the eggs of the insect.
Ex. Scrub typhus in thrombuculid mite
Trans stadial transmission:
The disease agent is passed from one stage
of life cycle to another and then transmit the
disease to man.
Ex. Typhoid in housefly
13. 13
General principles of arthropod
control
Protective measures:
1. Physical barriers: Mosquito nets, screening
of building against houseflies etc.
2. Chemical barriers: Application of repellent
cream to body and clothing. Ex. Use of
odomas against mosquitoes
14. 14
Aggressive Measures
1. Source Reduction (Environmental
manipulation): Ensuring efficient drainage
of waste water collection (to prevent
mosquito breeding) and quick and effective
removal of garbage to prevent fly breeding.
15. 15
2. Mechanical control – Use of sticky paper
bait for houseflies.
3. Chemical control – By use of insecticides
which are classified as Larvicides: against
Larvae.
Imagicides: against adults.
16. 16
They are also classified as:
Stomach poisons: Eg. Paris green for
anopheles larva and Baigon bait for
cockroaches.
Asphyxiants: Eg. Oiling against anopheles
larva, Fumigants (gases) like HCN, Se2 in
warehouses.
17. 17
Contact insecticides
Space sprays (knock down agents) e.g.
Pyrethrum
Residual sprays
Chlorinated compounds e.g. DDT
Organophosphorous compounds
e.g. Parathion, Malathion, etc.
Carbamates e.g. Carbaryl, etc.
18. 18
4. Biological Control – e.g. use of Gambusia
and Guppy fishes to eat anopheles larvae
5. Genetic Control – Releasing sterilized male
insects in such numbers that they compete
with the wild male insects to fertilize the wild
female insects and thus eventually reduce the
insect population. E.g. use of (irradiated)
sterile male Culex in control of filariasis.
19. 19
Mosquitoes
In Kenya, mosquitoes constitute the most
important group of insects of public health
importance. A study of them is done under the
following heads:
1. Structure and life history in brief.
2. Differentiation of mosquitoes.
3. Outline of mosquito-borne diseases.
4. Mosquito control measures.
5. Methods of collection and identification.
20. 20
Structure and Life History
Morphology: The body of a mosquito is
divided into head, thorax and abdomen.
Head: semiglobular, bears a pair of
compound eyes, a pair of antenna (bushy
in males), a pair of palpi and a central
piercile proboscis.
Thorax: round, has a pair of wings dorsally
and 3 pairs of legs ventrally.
Abdomen: long and narrow, 10 segments,
the last 2 fused to form genitals.
21. 21
-Has 4 stages – egg, larva, pupa and adult.
-The eggs are laid only in water and hence
presence of water is essential for the
existence of all mosquitoes.
- The metamorphosis is complete, the egg
and pupal stages are the resting stages while
the alternating larval and adult stages are
highly active.
Life History:
22. 22
The Life Cycle:
- Normally of about 2 weeks duration (Egg: 1-3
days, Larva: 7-10 days, Pupa: 1-3 days. Adult
life span about 2 weeks-1 month).
- Only the females are blood sucking and
males live on plant juices.
- Normally the mosquitoes (except Aedes)
have a flight range of ½ to 1 mile and this is
an important factor which determines the
extent of the area to be included under
mosquito-borne disease control operation.
23. 23
Differentiation of Mosquitoes
Genus Important
Species
Area of
Distribution
Anopheles Culicifacies
Philippinensis
Fluviatilis minimus
Sundaicus
Stephensi
Plains
Foothills
Coastal areas
Culex
Aedes
Mansonoides
Fatigans
Tritaniorrhynchus
Aegypti
Annulifera
Uniformis
24. 24
Anopheles Vs. Culex
Stage of Life Anopheles Culex
Egg - Boat shaped
- Laid singly
- Have lateral floats
- In fresh water
- Oval
- Laid in rafts
- Without lateral floats
- Usually in still dirty water
Larva -Lies horizontally with surface
of water
- No siphon tube
- Has palmate hairs on
abdomen
-Lies at an angle with the
surface of water
- Siphon tube +
- No palmate hairs
Pupa - Breathing trumpet (siphon
tube)
- Short and broad (funnel
shaped)
- Long and narrow (trumpet
shaped)
Adult -Body: at an angle with
surface on which it rests
- Wings: usually spotted
- Palpi of male: club shaped
- Palpi of female: as long as
- More or less parallel with the
surface on which it rests
- Non-spotted
- Not club shaped
- Shorter than proboscis
25. 25
Outline of Mosquito-Borne Diseases
Mosquito Disease
transmitted
Extrinsic
Incubation
Period
Mode of
Transmission
Causative
Agent
Reservoir of
the Agent
Anopheles Malaria Not less than
10-15 days
Bite of
infective
female
mosquito
Plasmodium:
vivax,
falciparum,
malariae &
ovale
Man suffering
from Malaria
Culex
fatigans Urban filariasis 10-14 days Deposition of
infective larva
at the site of
bite by female
mosquito
Wuchereria
bancrofti
Man
harbouring
microfilaria
Mansonoides Rural filariasis 10-14 days Deposition of
infective larva
on the site of
bite by female
mosquito
Brugia malayi Man
harbouring
microfilaria
26. 26
Mosquito Control Measures
Protective (Defensive) Measures:
Use of mosquito nets: The number of
holes/sq. inch is 150
Screening of buildings with wire mesh
having 16 meshes/inch
Use of chemical repellents like deet (diethyl
toluamide), dimethylphthalate, etc.
27. 27
Aggressive Measures:
Anti-larval measures:
Environmental Control:
It involves chiefly the water management. If
Anopheles are a problem, their breeding places
should be abolished by proper methods like
filling and drainage.
If Culex are a problem, the waste water
collected to be suitably drained.
In case of Aedes, the artificial collection of
water in containers, etc. to be eliminated and
prevented.
For Mansonia, the aquatic plants should be
destroyed and further growth checked.
28. 28
Chemical Control:
The commonly used larvicides are:
Mineral oil (Malariol) acting as asphyxiant applied
at the rate of 40-90 litres/hectare;
Paris green (copper acetoarsenite) acting as a
stomach poison is applied as a 2% dust at 1
kg/hectacre;
Synthetic insecticides baytex (Fenthion) 1000
applies as emulsion at 150-500 litres/hectare
Biological Control: By use of larvae eating (larvivorous)
Gambusia and guppy fishes in control of Anopheles
29. 29
Anti-adult measures:
Insecticides: As space sprays (knock-down
agents) – use of Pyrethrum extract (1 oz/1000 cu.ft.
space); ULV (ultra low volume) fogging of malathion
or fenthion in outdoors in control of Aedes (a day
biter). Residual sprays for indoors, lindane (0.5
g/m2), DDT (1 or 2 g/m2), malathion (1 g/m2), etc.
Genetic Control: Sterile males release technique
(Culex fatigans control) and other approaches like
use of insect growth regulators, chemosterilants,
etc.
30. 30
Methods of Mosquito Collection
and Identification
For Larva:
- A laddle (long-handled spoon) of 3.5” diameter
is taken and dips are made along the edges of
breeding water source.
-At least 5 dips are taken in each breeding place.
The number of larvae/dip are counted and
identified (using dissecting microscope) and the
average number of the five dips is taken as Larval
Density, an indicator of magnitude of mosquito
breeding in the area.
31. 31
For Adult:
The most commonly occurring adult Culex and
Anopheles mosquitoes rest in the day in darkness on
damp walls and corners of the room.
They are collected by using a suction tube – a glass
tube of 9” size is taken.
A gauze piece is tied on the one end and connected to a
rubber tube. The other end is kept open to suck in the
mosquitoes.
The adult mosquitoes are caught with the help of this
sucking tube from the dark corners of the living rooms,
cattle sheds, etc., by using torch light in the early hours
of the morning.
32. 32
The mosquitoes so collected are transferred to a test
tube which is subsequently closed with a cotton plug.
The mosquitoes collected are killed by vigorous
shaking of test tube. The total time of collection per
person is noted.
The mosquitoes collected are counted and
subsequently identified with a hand lens or dissecting
microscopes.
The total number of mosquitoes collected per person per
hour is calculated and expressed as Man Hour Density
(MHD). The mosquito catching is done for 2-3 days and
the average is taken for better reliability of the result.
The MHD of mosquito indicates the magnitude of
mosquito menace in the area.
34. 34
Housefly (Musca
domestica)
The presence of housefly is an index of poor
sanitation.
Structure:
-The body is divided into head, thorax and
abdomen, and it has 3 pairs of legs and 1
pair of wings.
-The legs and body are covered with
numerous short and stiff hairs, called the
tenent hairs, which contain a sticky
substance.
-Each leg has a pair of foot pads which help
the fly to walk on smooth surfaces.
38. 38
Life History
The female lays eggs in moist decaying organic
matter like human & animal excreta, manure heaps &
garbage.
The eggs are white, 1 mm size and just visible to
naked eye (resembling polished rice grain).
The eggs hatch larvae (maggots) which are creamy
white, segmented & footless, active and eat
voraciously & grow up to 1 cm. They resent light
and bury themselves under manure heaps.
When about to pupate, they migrate to outer dry
regions. The pupa measures ½ cm and is dark
brown and barrel-shaped.
From the pupa, adult fly comes out and the life cycle
is completed in 1-3 weeks.
39. 39
Mode of Disease Transmission:
They carry the pathogens
(mechanically) on their feet and hairy
legs. In addition, the flies frequently
vomit (vomit drop) and defecate which
also contain ingested pathogens in
viable state.
41. 41
Fly Control Measures:
Environmental control: Safe and
sanitary disposal of: human excreta
(sanitary latrines) & animal excreta
(manure pits with earth cover or by
composting); Garbage & refuse (refuse
bins with tight lids, composting, etc.). A
clean house with a clean surrounding is the
best solution.
42. 42
Protective measures: Use of fly-proof
meshes (14/inch) for windows and even doors
(provided with springs to keep always closed)
Mechanical control: By use of sticky fly
papers, fly traps, etc.
Chemical control:
Larvicides: Use of DDT, 0.5% Diazinon, etc. for fly
breeding sources
Imagicides: Use of baits (Baigon-2% propuxur) and
occasionally space sprays (Pyrethrum/Malathion)
43. 43
Lastly a “Fly Consciousness” should be
created among people through Health
Education and only with the active
cooperation of the people the fly menace
can be controlled.
44. 44
Sandfly (Phlebotomus)
These are slightly smaller than mosquitoes, light
brown-colored, hairy, winged insects with slender
long 3 pairs of legs.
The wings are held at 45° to body and these insects
(though winged) only jump (hop) short distances.
They mostly breed in cattle sheds, stables and
poultries and are within 50 yards from their breeding
place.
They are nocturnal biters (only female bite) and
during daytime hide in cracks & crevices of walls dark
nook corners of cattle sheds, poultry, bathroom, etc.
The life cycle consists of egg, larva, pupa and adult
and this is usually complete in 2-4 weeks, mostly in
cattle sheds and poultries.
50. 50
Control Measures
By locating cattle sheds, poultries and stables
50 yards away from house
By tight cement plastering of cracks and
crevices in the walls & floors
By use of insecticides like DDT spraying (1-2
g/sq. m.) of cattle sheds and other resting
places
Use of insect repellants
Sandfly net: 45 mesh/inch may be used to
protect against bite
51. 51
Tsetse Flies (Glossinae)
These are blood sucking insects found in
Africa and attack man and animals during
daytime.
Tsetse flies are vectors of Trypanosomiasis or
sleeping sickness, affecting man, domestic
animals and wild game.
They breed in woodland vegetation and
females give birth to larvae (no egg laying).
Control measures include aerial spraying of
insecticides of large areas for woodland
vegetation.
53. 53
Blackflies (Simulidae)
These are vectors of Onchocerciasis in
Africa and Central and South America.
They breed under submerged stones
and weeds in running streams.
As they have a flight range of up to 100
miles, the control measure is aimed at
aquatic breeding source through aerial
spraying of insecticides.
54. 54
Insecticides
Definition :
-An insecticide is a chemical used for killing
arthropods.
- A larvicide is an insecticide againist immature
stages of arthropods.
- Imagicide (adulticide ) is againist the adult
stages of arthropod.
55. 55
Classification
Chemical classification:
Group I : Organochlorine compounds –
DDT, Gammaxane.
Group II :Organophosphorous compounds
- Malathion, Fenthion, Fenitrothion,
Parathion.
Group III : Carbamates - Propoxur,
Carbaryl
56. 56
According to their mode of action:
Stomach poisons: These must be ingested
to kill ( effective against insects with mouth
parts for chewing) the arthropod. These
may be
Spread over the natural food as a spray or dust
( paris green againist anopheles larvae ) or it
may be incorporated in an attractive bait ( 2%
carbamate in baigon bait againist houseflies
and cockroaches ).
57. 57
Asphyxiants:
1. Fumigants : These are gases which asphyxiate
the arthropods. These are used in grain storage
bins, warehouses and other enclosed areas
where the insects cannot be reached by ordinary
sprays or dusts, or where such insecticides may
harm the material being infected.
Ex: Use of Hydrocyanic acid ( highly poisonous, to
be operated by experienced persons with gas
mask ). SO2 and Carbon di sulphide ( highly
flammable and explosive ).
58. 58
2. Oiling:
Ex. Malariol is applied on the water surface
which forms a thin layer and kills larvae and
pupae of mosquitoes.
59. 59
Control insecticides:
which kill insects primarily by contact.
1. Knock down agents : these have a rapid paralysing
effect . Ex: Pyrethrum.
2. Residual agents : are placed directly on or some
surface upon which the arthropod will walk or rest and
where it may retain its toxicity for 3-4 months. Ex:
DDT, Malathion, etc. in anopheles control.
3. Space sprays: insecticidal solutions which are
dispersed as finely atomized droplets by means of a
spraying device. Ex. Pyrethrum against mosquitoes.
60. 60
Contd..
4. Aerosols : a modified space spray where the
liquefied gas aerosol consists of a solution
of insecticide in dichloro difluoro methane of
similar material which forms a gas at
ordinary room temperature. Ex.Pyrethrum
and DDT against mosquitoes
61. 61
5. Ultra low volume ( ULV ) fog :
Application of highly concentrated of
undiluted insecticides in the fornm of a fog
– ( using helicopters or aeroplane ) Ex.
Malathion in Aedes Aegypti control.
62. 62
Selective insecticides
1. DDT ( Dichloro-Diphenyl-trifluoroethane )
It is a white e amorphous powder. The
technical DDT contains 70-80% Para para
isomer which is a active ingredient.
It is a contact nerve poison.
It permeates through the cuticle and causes
paralysis of the nervous system.
The commercially available water dispersible
DDT powder contains 50% or 75% DDT.
63. 63
The DDT ifs taken in bucket, the measured
quantity of water is slowly added to it and
stirred to prepare a solution.
A 5% suspension when sprayed at a rate of 1
gallon/1000 sq.feet gives a dosage of
200mg/1000sq.feet.
As a residual spray it is sprayed indoors on
the walls, ceiling etc. and it remains effective
for 3 months.
As a 10% dust, DDT is used against lice, fleas
and ticks.
64. 64
2. BHC (Benzene Hexa Chloride )
it is a white or chocolate powder with a
musty smell. The technical BHC contains
13-16% of gamma isomer.
Pure BHC containing 99% of gamma isomer
is called Lindane.
The commercially available powder contains
50% of the BHC.
It is a residual nerve poison.
65. 65
3. Malathion
It is the least toxic of all
organophosphorous componds and is used
as an alternative to DDT in some areas.
Commercially water dispersible powder is
available and the same is used in doses of
100-200mg per sq.foot
It is also widely used as ULV spray.
66. 66
4. Pyrethrum
It is extracted from the flowers of
crysanthemum.
Its active ingredients are pyrethrins/
It is used as a knock down agent and it has
no residual action.
67. 67
5. Malariol
Applied at 15-20 gallons per acre of water
cuts off supply of air to larvae and pupa of
mosquitoes by forming a thin film over the
surface of water.
However it makes water unsuitable for
domestic purpose and fishes may be killed.
68. 68
6. Paris Green (Copper aceto arsenite )
it is a green crystlline powder.
When sprayed as a 2% dust, at the rate of
1kg of actual paris green per hectare of water
, it kills the anopheles larva by acting as a
stomach poison.
In this dosage it does not harm fish, man or
domestic animals.
69. 69
Application of insecticides
Spraying
Knock down agents : Pyrethrum
Residual sprays : DDT, BHC, malathion
Space sprays : Pyrethrum
Aerosols : Pyrethrum and DDT
ULV fogging : Malathion
70. 70
Dusting
Individuals : 10% DDT in body louse infestation
Rodent burrows, holes : 10% DDT in rodent
and rat flea control.
Fumigation
Hydrocyanic acid against rat fleas, bugs,etc.
71. 71
Repellants
A repellent is a chemical applied to the
skin or clothing or other places to
discourage arthropods from sitting on
and attacking an individual.
1. Creams : Dimethylphthalate against
mosquitoes, sand flies, ticks and mites.
2. Fumes (vapours) : mosquito coils
72. 72
Insecticide resistance
The development of an ability in strain of
insects to tolerate doses of toxicants which
would prove lethal to the majority of
individuals in normal population of the same
species.
it may be due to
Genetic factors - mutation
Biochemical factors - toxicant is converted
to a nontoxicant by body enzymes.
73. 73
Types
Single resistance : against only one
insecticide
Double resistance : against two insecticides of one
group Ex. DDT and Gammaxane
Cross resistance : against insecticides of two
different groups – carbamates and OP
compounds.
