Diagnostic Microbiology Flashcards
What are the objectives of Diagnostic microbio-
Confirm the diagnosis, Guide in treatment management
What are the points to consider for collecting a sample- Collection time period-
3rd week after infection in thyphoid, Handling and transport medium
What are the most common specimen types-
Blood, CSF, urine, Respiratory secretions, NP swab (COVID), feces, Genital secretions, Exudates and biopsy
What are the certain cases when blood is collected as a sample-
Hyperacute infection, G -ve sepsis and Subacute endocarditis- 3 samples in 24 hours
What are the steps to collect blood- Sterile syringe, tourniquet to expose veins, prepare the site-
2% iodine, 70% alc. And draw 20ml.
How long should the sample be incubated for Brucella infection-
37 deg, for 5-7 days
What are the criteria for diagnosing Bacteremia-
Growth of the same organism at different sites of sample
Criteria for diagnosing contamination-
Different bottles and different cultures at different times
What types of samples are etiologically significant?
- S. viridans in endocarditis, G +ve; E. coli- sepsis, G -ve
How to collect samples for CNS infections?-
LP and CSF culture, Cell count, Protein
For a patient presenting with COVID-19 like symptoms, how to collect a sample?-
Throat swab/throat washing and Nasopharynx swab in charcoal for COVID-19 and Pertussis
If a patient presents with symptoms of LTRI, how to collect the sample?-
Wide mouth plastic sterile cont, before therapy, in the morning, Good sputum with WBC, saliva, >25 cells, bad sample.
What are the other ways to\collect LTRI samples-
Transtracheal aspirates, Bronchoscopy, Lung biopsy, and Bronchoalveolar lavage.
How are samples collected for an Eye infection?-
Sterile platinum loop, smooth, thin plastic/glass rod, Stuart medium swab, amd infection around eyes.
For a patient with an earache, what are the ways to collect a sample for diagnosis?-
Swabs
What are the various ways to collect samples for GIT infections?-
Feces- into a sterile bedpan, 1-2 ml, add 6ml glycerol to help transporting and rectal swab.
In the presence of an exudate- wounds, and abscess, how are samples collected?-
Pus from an abscess and open wounds, In closed spaces, Pleural or peritoneal or synovial surfaces aspiration.
What is the term for the wide open wounds?-
Carbuncle and cellulitis can still be aspirated.
What diseases constitute Upper UTI?
Kidneys- Leptospirosis, typhoid, and AGN
When to “catch” urine specimen for men?-
Clean meatus, midstream
Urine sample collection for a female-
Spread the labia and vulva, wash it thoroughly and collect midstream.
If suffering from urinating issues what method should be implemented?-
Suprapubic.
How is vaginal infection confirmed?
often purulent,
putrid or fishy-smelling
In cases of vaginosis, candidiasis, and trichomoniasis
Swab on the
cervical surface and not on the
vaginal wall.
Syphilis:
Try to irritate the surface of the wound,
the Chancre, with acetone or ether. Then,
observe the development of some fluid coming
out, which is the one collected and used for examining Treponema pallidum. Organisms will
be inside the lesion.
ANAEROBIC INFECTIONS
The large majority of human normal flora are
anaerobes, and when they are displaced from
their normal sites, they produce disease.
CHARACTERISTICS / CONDITIONS WHICH
SUGGEST ANAEROBIC INFECTIONS
SUGGEST ANAEROBIC INFECTIONS 1. Often contiguous with the mucosal surface 2. Tend to involve many organisms, which these organisms are the anaerobes plus the aerobes; a multi-etiology. 3. Tend to form closed-space infections either as discrete abscess (e.g. Lungs, brain, pleura, peritoneum, pelvis) or by burrowing through tissue layers 4. Pus with a foul odor 5. Favored by the reduced blood supply
Collection of specimens for anaerobic
○ Swabs are not used ○ For closed abscesses, aspirate aseptically, avoid entry of air by sealing the needle after the procedure ○ Use appropriate transport media when the specimen has to be brought to a distant laboratory.
THE 5 I’S OF CULTURING MICROBES
Inoculation, incubation, isolation, Inspection, and Identification
Media -
providing nutrients in the laboratory
Most commonly used media
○ Nutrient broth - liquid medium
containing beef extract & peptone
○ Nutrient agar - solid media containing
beef extract, peptone & sugar
Agar -
- a complex polysaccharide isolated from
red algae. - Solid at room temperature, liquifies at
boiling (100℃), does not resolidify until it
cools to 42℃) - Provides a framework to hold moisture &
nutrients - Not digestible for most microbes
Forms of physical Media
liquid, semisolid and solid
Types of media
Synthetic- pure inorganic or organic
Complex/non-syn- non chemically defineable ingredient
General-purpose media - grows a broad range
of microbes, usually nonsynthetic
Enriched media - contains complex organic
substances such as blood, serum, hemoglobin or
special growth factors required by fastidious
microbes
ENRICHED MEDIA
● The most common enriched media is blood agar.
● The organism in plate A produces hemolysis
● Chocolate agar is a variation of blood agar plate.
It is blood agar which is heated, it turns to a brown
color.
Selective Media
● Contains one or more agents that inhibit growth
of some microbes and encourage growth of the
desired microbes
● Example: Salmonella/Shigella agar medium- will
only allow the growth of Salmonella species and
Shigella species
Differential media
● Allows growth of several types of microbes and
displays visible differences among desired and
undesired microbes
● A good example of this is the MacConkey agar. It
will allow the growth of organisms that can
ferment lactose and organisms that do not
ferment lactose
misc. media
Reducing medium - contains a substance that
absorbs oxygen or slows penetration of oxygen
into medium; used for growing anaerobic bacteria
Carbohydrate fermentation medium - contains
sugars that can be fermented, converted to acids,
and a pH indicator to show the reac
Magnification
ability to enlarge objects
Resolving power -
ability to show detail; the
higher the resolving power, the higher the ability
to see the details of the particular object being
examined
Oil immersion lens
● The oil makes it possible for the organism being
examined to be seen more clearly in the
microscope.
● The rays of light are concentrated to the point of
examination and bounce back to the eyes.
Without oil, you lose some of the light waves.
Bright- field -
The most widely used, the specimen is
darker than the surrounding field
Dark Field-
brightly illuminated specimens
surrounded by darkfield; a special type of light
microscope whereas instead of seeing the field to
be brightly illuminated, what you will see is a
background of black and only the ones that you
want to see are illuminated. So they will stand out.
This is the one used for Treponema pallidum and
also for Leptospirosis
Phase-contrast -
transforms subtle changes in
light waves passing through the specimen into
differences in light intensity, best for observing
intracellular structures
FLUORESCENCE MICROSCOPY
Modified compound microscope with an
the ultraviolet radiation source and a filter that
protects the viewer’s eye
Useful in diagnosing infections-For example, Treponema pallidum (Syphilis), you
can tag the antibodies against the T. pallidum
with special dye
ELECTRON MICROSCOPY
Forms an image with a beam of electrons that can
be made to travel in wavelike patterns when
accelerated to high speeds.
● Electron waves are 100,000X shorter than the
waves of visible light.
Two types of electron microscopy
○ Transmission electron microscope (TEM) - transmits electrons through the specimen; darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts ○ Scanning electron microscope (SEM) - provides detailed three- dimensional view. SEM bombards the surface of a whole, metal- coated specimen with electrons while scanning back and forth over it.
STAINING PROCEDURES
● In bacteriology, staining procedures are very
important because one staining procedure gave
us a very big classification of the bacteria. The
Gram staining classified the bacteria into two
types of microorganism– the gram positive
organism and the gram negative organism. Their
cell walls are responsible for their classification.
Wet mounts & hanging drop mounts -
allow examination of characteristics of live cells:
motility, shape, & arrangement; you examine the
specimen as is
Fixed mounts
are made by drying & heating a
film of specimen. This smear is stained using
dyes to permit visualization of cells or cell parts.
Dyes
○ Cationic dyes - basic, with positive
charges on the chromophore
○ Anionic dyes - acidic, with negative
charges on the chromophore
○ Positive staining - surfaces of microbes
are negatively charged and attract basic
dyes; the cell wall will be stained
○ Negative staining - microbe repels dye
& it stains the background; an example of
this is when you want to see the
capsulated microorganism,
Simple stains -
one dye is used
Differential stains
use a primary stain and a
counterstain to distinguish cell types or parts. Gram stain Acid- fast stain
Special Stain
- India ink capsule stain of Cryptococcus neoformans. You use the India ink so what you see is the Cryptococcus’ very thick capsule. - Flagellar stain. The red ones are the flagellar stains
