عملي ميكرو ترم 1

1. 2019
MAHMOUD ESSA

2. Sterilization ....&disinfection
Revision

4. Q1/Identification? bacteriological loop
Q2/Uses? 1-planting techniques of bacteria
2- direct smear
Q3/methods of sterilization to it ?
1-direst flame
2-autoclave
3-hot air oven
Q4/Difference between sterilization &disinfection ?
1-sterilization : killing of all living forms of microbes including
Bacterial spores .
2-disinfection : destruction or inactivation of most but not all
Pathogenic forms of microbes ; but not including bacterial spores
.
Q5/ levels of disinfection ?
1-High Level disinfection
-In this level most microbes are killed except some spores
-Chemicalstereliants achieve this level
2-Moderate Level disinfection
-In this level spores are not killed at all but M.Tuberculosis is killed
3-Low Level disinfection
-In this level M.tuberculosis is not killed.
Q6/ Monitoring of dry heat ?
Bacillus atrophaeus

6. Q1/identification ?
Hot air oven
Q2/type of sterilization ?
Dry heat
Q3/ Time require for sterilization ?
1-160 °C for 2 h
2-170 °C for 1 h
Q4/Objects for sterilization in it ?
*glassware, oils, powders , metal instruments
Q5/monitoring ?
By bacillus subtilis

8. Q1/identification?
*incinerator (dry heat)
Q2/ uses?
*destruction of contaminated material

10. Q1/Identification ?
*pasteurization system (moist heat)
Q2/ Uses ?
•Pasteurization of milk & kill bacteria specialy
•mycobacterium tuberculosis
•brucella abortus
•Salmonella
•Q3/ mechanism of action ?
Types;
1-Holding past.;
Heating for 30 min at 63°c
2-Flash past.;
Heating for 20 sec at 72°c
BOTH are followed by rapid cooling below 10°c

12. Q1/Identification ?
Autoclave (steam jacketed autoclave)
Q2/Type of sterilization ?
Moist (steam) sterilization above 100 °c
Q3/uses?
*Sterilization of culture which not damaged by heat.
•Bed linen. But not plastic instruments .
Q4/Temp & Time require? …Mechanism of
Action?
* 121°C at double atmospheric pressure for 20-30 min.
*134°C at triple atmospheric pressure for 3-6 min.
….* fluid under pressure with high temp increase boiling
Point more 100°c used for sterilization .

14. Q1/identification ?
Autoclave (Flash)
Q2/ Uses?
•emergency cases in operation for sterilization
•of metal instruments

16. Q1/Identification ?
plasma gas sterilizer
Q/ type of sterilization ?
Gaseous chemical sterilization by
(steam of hydrogen peroxide)
*Other method of same type (ethylene oxide )
Q3/ uses ?
*sterilization of long narrow lumen instrument as
laparoscope &arthroscope
Q4/ Advantages?
*non toxic
*not require long time .unlike Ethylene oxide

18. Q1/Identification ?
biological indicator for monitoring efficiency of autoclave .
Q2/Bacteria used in biological indicator ?
bacillus stearothermophilus spores
Q3/Other methods use for monitoring efficiency
of autoclave ?
*mechanical (recording the temperature, time and pressure)
*chemical ( chemical indicators change its color under certain temp)
*biological (geobacillus stearothermophilus spores)

20. Q1/Identification ?
Vacuum filter .
Q2/Type of sterilization ?
Filtration (mechanical method )
Q3/uses?
Filtration of large volume of fluid

22. Q1/Identification ?
High-efficiency particulate arrestance (HEPA)
(air filter) one of filtration method of .ster .
Q2/Uses?
1-filtration of air in operation room &
drug filling cubicles
Q3/method of monitoring?
1- fungus aspergillus
2-endopigment producing serratia marcsens

24. Q1: Identification ?
Surgical instrument
Q2:Methods of sterilization ?
1-autoclave
2-plasma gas sterilizer
3-Ethylene oxide

26. Q1: Identification ?
Membrane (millipore) filter .(filtrarion method)
Q2:uses?
1-filtration &sterilization of drug
2- filtration &sterilization of biological fluid.

27. Notes
Sterilization:
1-Dry heat
2-Moist heat only (autoclave)
3-Gaseous chemical sterilization
*ethylene oxide
*plasma gas sterilizer
4_radiation
5_filtration
6_chemical sterilization (sterilants)
•hydrogen peroxide.
•* glutaraldehyde .
•Peracetc cid
Disinfection :
1-Moist heat (pasteurization )
(boiling )
2_UVrays
3-chemicaldisinfection

28. Antigen antibody reaction

30. Q1/Identification?
*direct agglutination (slide agglutination )
Q2/Application ?
Blood grouping
Q/Other methods of Antigen antibody reaction?
1-pricipetation
2-ELISA
3-neutralization
4-immunofluorescence
5_complement fixation

31. Titre

32. Q1/Identification ?
tube agglutination method
Q2/Type?
Agglutination (antigen antibody reaction )
Q3/Application ?
1_zone phenomenon
2_dignosis of enteric fever &typhus fever
3_Widaltest for diagnosis of salmonella
Q/definition?( titre of antiserum)(zone phenomenon)?
1_highest dilution with low concentration showing clearly
Visible agglutination ..
2_observable union between antigen&antibody occurs best
When both reactants are present in optimalproportion …with
No excess amount of any of reactant .

33. 1
2
3
1_Toxogenic diphteria
2_unknown
3_ non _Toxogenic diphteria

34. Q1/ identification ?
Elek,s test .
Q2/type ?
Precipitation Ag &Ab reaction (double diffusion)
Q/Application ?
Detection of the pathogenicity (toxin ) of diphteria ..

36. Q1/identification ?
*precipitation in (agar gel )..Single diffusion … Ag&Ab reaction..
Q2/Uses ?
* Estimation of concentration of (I g )

38. Q1/identification?
Double diffusion in agar gel
Q2/ USES??
*Grouping streptococci .

40. Q1/Identification ??
flow cytometry machine ..
Principle?
rapid analysis of multiple characteristics of single cells. That
measures optical and fluorescence characteristics of single cells
&Physicalproperties,
Q2/Application ?
1-enumeration of lymphocyte subpopulation
In HIVvirus patient or in transplantation ..
2-tumor cell phenotyping
3_rapid detection of virus , fungus &virus

42. Q1/Identification ?
Immunochromatography
Q2/type ?
Latex agglutination ..
Q3/Application?
_Diagnosis of pregnancy
_ Diagnosis of hepatitis

43. 2019

44. 2019
MAHMOUD ESSA

45. Diagnostic molecular
biology method

47. Q1/Identification ?
Thermalcycler
Q2/Reaction used in it ?
polymerase chain reaction (PCR)
Steps of (PCR)
A- Nucleic acid extraction.
B- Nucleic acid amplification (Thermal cycle).
C- Detection of the amplified nucleic acid.
Q3/ Steps of reaction (thermal cycle)?
1-Denature DNA
The DNA is heated to 95°C. This breaks the weak hydrogen bonds that hold DNA strands
together in a helix, allowing the strands to separate creating single stranded DNA.
2-Primer Annealing
The mixture is cooled to anywhere from 40-60°C. This allows the primers to bind (anneal)
to their complementary sequence in the template DNA.
3-primer Extension
The reaction is then heated to 75°C, the optimaltemperature for DNA polymerase to act.
DNA polymerase extends the primers, adding nucleotides onto
the primer in a sequentialmanner, using the target DNA as a template.
Q4/Types of PCR ?
*ASYMMETRIC PCR *Allele-specific PCR
*quantitative PCR *RACE PCR

49. Q1/Identification ?
polymerase chain reaction (PCR)
Q2/Steps?
A- Nucleic acid extraction.
B- Nucleic acid amplification (Thermal cycle).
C- Detection of the amplified nucleic acid.
Q3/Application of PCR ?
1) diagnosis of infections:
a-Non cultivable organisms.
b-Pathogen in tiny quantities.
c- Rapid diagnosis of slowly growing organisms.
d- Identification of pathogenic species.
e- Identification AB resistant strains.
2)Diagnosis of cancer
}Detection of latent virus in neoplastic cells.
}Early diagnosis by detecting genetic changes.
3)Diagnosis of inherited diseases.
4)Organ transplantation.
5)Forensic medicine.
6)Basic biological & medical research.
Q4/ other methods of amplification ?
*ligase chain reaction (lCR)
*branched DNA *nucleic acid sequence based amplification

50. Q1/methods to identify of DNA arising from amplification ?
1-hybridization by probe
2-agarose gel electrophoresis
3-sequencing

52. Q1/Identification?
agarose gel electrophoresis
Q2/ Application?
*To identify of DNAarising from amplification(PCR
•To identify …
•(restriction fragment length polymorphism )

53. Biochemical
reactionS

54. PO
S
Neg

55. Q1/Identification?
Indole production test.
Q2/Media used?
Peptone water
Q3/Chemicalreagent used?
Kovac,s reagent
Q3/Principle?
Decomposition of tryptophane existed in peptone to indole
By certain bacteria ..(E.coli) (indole positive )
2 1
1-positive
2-negative

56. Q1/Identification ?
Citrate test
Q2/Indicator ?
thymol blue indicator
Q/Principle ?
Certain bacteria use citrate as a source of carbon
and energy ,producing sodium bicarbonate which
convert media into alkaline that Convert indicator
to blue color ..
As (Klebsiella pneumoniae)

57. Q1/Identification ?
IMVC test
Q2/Bacteria produce this result ?
E.Coli
Q1/Identification ?
IMVCtest
Q2/Bacteria produce this result ?
Klebsiella

58. methylred test ?
*glucose phosphate peptone media.
*fermentation of glucose producing acid media .
*detection of acidity by methyl red (indicator) convert to red color
vogus proskauer test?
*glucose phosphate peptone media.
•fermentation of glucose producing acetylmethylcarbinol (acetoine)
•react
•With potassium hydroxide (indicator)give red colour ..
0

59. coagulase test (slide method)
Q/Application ?
differentiation between types of
staphylococci.
*staph aureus (+ ) producing
coaglulase enzyme
•staph epidermidis (-)
•* staph saprophyticus (-)
coagulase test
(tube method)

60. Urease test
H. pylori secrete the urease enzyme..
conversion of urea to ammonia ….
which raises the pH of the medium
and changes the color of phenol red (indicator)

61. Q1/Identification?
Catalase test
Q2/Principle ?
Bacteria produce catalase enzyme dissociate H2O2 to H2O +O2
Q/ Application ?
differentiation between staph..(+) &sterpt (-)

62. Q/identification ?
Oxidase test
Q/Idea of action ?
Certain bacteria produce oxidase enzyme lead to destruction oxidase rea
Producing purple color ..
Q/Examples ?
* Helicobacter pylori
* Pseudomonadaceae
*Neisseria

63. Q/identification ?
dnase test
*certain bacteria produce dnase enzyme which destruct
Dna material in the media forming ring of precipitation ..
Q2/Application ?
Identify staph aureus .. (dnase postive )

64. Q/Identification ?
H2S production test
`
S=(Protus)
Q/examples ?
*some types of salmonella
•proteus bacteria

65. Q1/ identification ?
Analytical profile index (API)
Q2/Uses ?
Rapid diagnosis of infection
*API 20s for strept diagnosis

66. *Gram staining
_ Crystal violet (primary stain)
_ Iodine
_ Alcohol (decolorizing agent)
_ Safranin (counter stain)
ZZeeiihhll NNeeeellsseenn ssttaaiinn
__ fixation
_ Bunsen burner
Decolorization
acid-alcohol
Counter stain
Methylene Blue

67. Culture
media

68. *nutrient agar
*composition
} Nutrient broth +agar agar
*Sterilized in the autoclave
*Simple Solid media
*uses
_ Pseudomonas media (+)
(give green color) which is oxidase
BBlloooodd aaggaarr ::
*Enriched media
*composition
__nnuuttrriieenntt aaggaarr ++1100 %% bblloooodd aatt 5555 °°CC
**sstteerriilliizzaattiioonn iinn aauuttooccllaavvee
++ sstteerriillee bblloooodd
**iinnddiiccaattoorr tthhaatt ::
Complete haemolysis e.g. staph.
aureus &strept .pyogenes
(clear zone
around colonies)
In complete haemolysis e.g.
viridians streptococci &pneumococci.
(greenish zone around colonies)

69. **CChhooccoollaattee aaggaarr
*Enriched media
*Heated bl agar
*Prepared as bl agar but at 100 °C
*media for Neisseria &Haemophilus
Which is oxidase (+)
Remember

70. Lofflers serum
Enriched media
*diagnosis Diphtheria bacilli
*composition
3 parts of serum from animal
1part glucose broth

71. Lowenstien Jensen medium (L-J)
*Selective media
*For TB bacilli
*TBgrow after 2-8 weeks
•Beaten egg +mineral salts &
•malachite green

72. } TCBS: selective media
} vibrio cholera (Alk.media)
} thiosulphate , citrate &bile as
selective substances
} ,thymol blue &bromothymol give
yellow color in acidic Ph
} vibrio cholera produce yellow colonies

73. MacConkey medium
DDiiffffeerreennttiiaa mm eeddiiaa
*lFor enteric bacteria
Lactose fermenters ( pink colonies) E-coli
Non Lactose fermenters ( pale colonies) salmonella
&shigella

74. Mannitol salt agar
Differential media
isolation of staph.aureus from contaminated samples
} Staph aureus produce yellow colonies (+)
} Staph epider. (-)

75. Triple sugar iron agar
*Non carbohydrate fermenter (red slant &red butt)e.g. ps .aeruginosa
*Fermenter of glucose only (red slant &yellow butt)e.g salmonella&shigella
* Lactose &sucrose fermenter (yellow slant &yellow butt)e.g.Esh.coli &klebsiella

76. *anaerobic gas pack system
*cultivation for anaerobic
bacteria .As clostridium
** robertson cooked meat medium
* Anaerobic culture media
Component :
1-cooked minced meat
2-broth meat
3-reducing substance

77. Microscope

79. -Magnification power of light microscope
=magnification of the eye piece lens x
magnification of the objective lens
-Magnification of the objective lenses :
Low power lens : 10 times .
High power lens : 40 times .
Oil-immersion lens : 90 times .
-Magnification of the microscope when using the low power lens = 10 x 10 = 100 times .
-Magnification of the microscope when using the high power lens = 10 x 40 = 400 times .
-Magnification of the microscope when using the oil-immersion lens = 10 x 90 = 900 times
.
Resolution :
0.25 µ
Light microscope

80. Dark-ground microscope
It's used to visualize delicate organisms
as spirochetes of syphilis
.
fluorescent microscope:
The source of illumination is
ultra-violet radiation.

81. Electron microscope :
Magnification :
100000
Resolution :
1nm
Source of light ?
Electrom gun producing electrons

82. antibiotic
sensitivity
test

83. Q/Identification ?
antibiotic sensitivity test
*Disk diffusion method
Q/Other tests for antibiotic
sensitivity test?
*tube dilution method
*E- test
Q/Identification ?
tube dilution method
Q/Application ?
Get MIC
Q/Def?
Minimal inhibitory concentration (MIC) :
The tube with highest dilution with no visible
turbidity is the concentration that inhibit
bacterial growth .

84. Q/Identification ?
E-test (Epsilometer test)
Q/ Application ?
MIC :
The zone edge intersects the test
strep

85. 2019
Mahmou essa