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Practical 2 - Food Born Infections Part

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23 views35 pages

Practical 2 - Food Born Infections Part

Uploaded by

Raneem Mohammed
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Level 3

Semester 6
• Module (Digestive system and Nutrition module BMS304)
Practice Title

Methods of diagnosis of the


bacteria causing foodborne
infections (part 1)
Instructor information
• Contact: Professor Doctor Mohammed Mahmoud El-Naggar
• Department. Medical Microbiology and Immunology
• Official email: [email protected]
• Mobile (optional): 01126625177
• Academic hours:
➢ Sunday: 9:00-11:00 AM
Practice Content
• Method of stool sample collection.
• Sample examination.
• Laboratory diagnosis of bacteria causing food
born infections.
Learning outcomes
- By the end of this lecture the students will be
able to:

1. Name the different media required for


isolation of foodborne bacteria.
2. Interpret results of different laboratory tests
used to confirm diagnosis of food born bacteria.
Acute intestinal infection

1. Faeces .
– clean containers
– In case of delay transport
specimen in glycerol saline.
2. Rectal swab
3. Rectal tube in
(cholera) rectal swab
4. Bile by duodenal drainage in
cholycystitis.
5. Vomitus in food
poisoning.

Faeces
What are food born
bacteria?
1. E.coli
2. Salmonella & Shigella
3. Staphylococcus aureus
4. Brucella
5. Clostridium
6. Cholera
7. Helicobacter pylori
8. Campylobacter
Laboratory diagnosis of E.
coli:
1- Sample:
stool ‘some strains’
2- Direct Gram stained film:
 Gram negative bacilli, non spore forming ,motile
,most are non capsulated
3- Culture:
A- Culture chs:
O2 – CO2 – Temp.
B- Media:
- Ordinary media: Grow
- Indicator media: Mac Conkey’s →LF →Rose
pink

C- Colony identification:
- Colony characters
- Gram stained film from the culture
- B.Rs
Biochemical reactions:
Ferment: (glucose, maltose, mannite, lactose and sucrose) with acid and gas
production.
IMVC:

4- Typing:

- Antibiotic sensitivity → strain


- Serotyping: with ‘enteropathogenic antisera’
against O, H, K antigens

Ejkmann test: (E. coli)


Use: To differentiate fecal from non-fecal E. coli
Principle: Fecal strains of E. coli can grow on fluid MacConkey’s medium at 44°C
producing
acid and gas (Ejkmann positive).
Salmonella & Shigella
• Salmonella:
Gram negative
bacilli
motile
Non spore • Shigella: non
forming motile
Non capsulated
Diseases caused by Salmonella:

1-Typhoid fever (S. typhi, S. paratyphi A,B,C)

2- Food poisoning (S. typhimurium,


S. enteretidis)
Lab diagnosis: (Typhoid) case

1- Sample:
1st week: Blood
2nd week: Stool, serum
3rd week: Urine, serum

2- Direct Gram stained film:


3- Culture:
(1st week) Blood or clot culture - (2nd) Stool - (3rd)
Urine
A- Culture chs: O2 – CO2 – Temp.

B- Media:
- Ordinary media: grow
- Selective media: - SS agar →pale
- Indicator media: Mac Conkey’s, DCA→ Pale.
- Enrichment media: Selinite, tetrathionate broth
3- Culture:
Enrichment
▪ Selenite
broth
▪ Tetrathion
ate

Nutrient b) Selective
SS,HE,XL
agar Media D

Indicator
▪ Machonkey
▪ DCA
Salmonella on SS
media
C- Identification of obtained colonies
by:
a. Film stained by Gram stain: shows morphology
(Gram-negative bacilli, motile, non capsulated
and non sporulated).

b. Biochemical reactions:
• Ferment: glucose, maltose, mannite
• (S. typhi) → Acid (S paratyphi A,B,C) → Acid
& Gas
• Lactose is not fermented.
- Indole, Voges-Prosk., Urease tests: Negative
- H2S prod. from ‘thiosulfate’: Positive
S. typhi S. paratyphi
4- Serology: from 2nd week
- Slide agglutination
- Tube agglutination ‘Widal test’
(Widal tube agglutination test).
The patient’s serum is tested for its titres of antibodies against
H, O and Vi antigens.
• O antibody appears early and disappears early
• H antibodies appears late and disappears late
• So we can determine the onset of infection by interpreting
the titre of both O and H antibodies:
• If the O titre is higher than the H titre this means early
infection
• If the H titre is higher than the O titre this means late
infection
H-Ag H-Ag H-Ag H-Ag
O-Ag typhi paratyphi paratyphi
paratyphi
A B C
O Ag H Ag H Ag H Ag H Ag Result
typhi paratyphi paratyphi paratyphi
A B C
+ + - - - S. Typhi
inf.

+ - + - - S. P.typ. A
inf.

+ - - + - S. P.typ. B
inf.

+ - - - + S. P.typ. C
inf.

+ + + + + Recent
vaccine
- + + + + Old
vaccine
original sample

serum
total volum ( serum + diluent )
Tube agglutination ‘Widal test’

0.1ml
serum +
0.9ml
saline

titre: Reciprocal of highest dilution of the serum that


gives positive reaction.
* Rising titre is diagnostic especially in results with
low titre.
Laboratory diagnosis of Shigella
infection (Bacillary dysentery):

1. Specimen: stool which contains mucus and


blood.
2. Culture:
•Facultative anaerobe
•Media:
A liquid enrichment medium (selenite broth) is
inoculated with stool specimen and subcultured
into SS after a short growth period produce pale
non lactose fermenting colonies on MacConkey's
agar.
3. Identification of obtained colonies by:
• Film stained by Gram: to show morphology(Gram negative, non
motile, non-sporulated, non capsulated bacilli).

•Biochemical reactions:
Glucose Mannite Lactose
S. dyenteriae A − −
S. flexneri A A −
S. boydi A A −
S. sonnei A A Late
Laboratory diagnosis of Staphylococcus aureus
food poisoning:

1- Samples: stool, food remnants

2. Direct film stained with Gram

stain: for characteristic

morphology (Gram positive cocci

arranged in clusters, non motile,

non spore forming.


3. Culture

 Staphylococci are facultative anaerobes, optimum


temperature: 37C & It can grow in normal atmospheric Co2
concentration.
 Staphylococci can be grown on ordinary media
 On blood agar S. aureus cause  hemolysis .
 S. aureus can grow on mannitol salt agar producing (yellow
colonies).which is selective for its isolation , as S.aureus but
not other staphylococci ferment mannitol and salt inhibits
other normal flora but not S.aureus
 S. aureus produce golden yellow endopigments.
golden yellow endopigments of
Staphylococcus aureus on nutrient
B- haemolysis of Staphylococcus
agar aureus on Blood agar
4. Identification of colonies
by :
a. Film stained by Gram for characteristic
morphology .
b. Biochemical reactions :
All staphylococci are catalase positive.
(This test differentiate staphylococci from
streptococci which are catalase negative) .
S. aureus can be identified by being coagulase and
DNAase positive ,and by mannitol fermentation.
Positive tube coagulase test Catalase test
Diagnosis of staphylococcal
food poisoning:
• The food remnants, vomitus and stools are
cultured on mannitol-salt agar and the isolated
staphylococci are identified by their biochemical
activities, catalase, coagulase and DNase
production.
• To trace the source of contamination of food, the
food handlers are tested for the possibility of
being nasal or skin carriers of strains belonging to
the same phage type as the strain causing food
poisoning.
Phage typing

1-Plates are inoculated


with the test strain, and
dried.
2-A drop of each phage is
placed in a marked
square area.
3-After incubation, lysis
will occur in the areas
of the corresponding
phage type.
References
• Cheesbrough M. (2000): Microbiological tests. Cited by
Cheesbrough, M., (ed.) District Laboratory Practice in
Tropical Countries, Part 2, Microscopical techniques used in
microbiology, Cambridge University Press, UK.

• Koneman E W, Allen S D, Janda W M, Schreckenberger R C,


Winn W C, Procop G W, et al. (2006). Introduction to
microbiology. Part II: Guidelines for the collection,
transport, processing, analysis, and reporting of cultures
from specific specimen sources. In Koneman EW, Allen SD,
Janda WM, Schreckenberger RC, Winn WC, Procop GW, et
al. (Eds.), Color atlas of diagnostic microbiology (6th ed.,
pp. 67-110). Lippincot, Philadelphia.

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