Culture Dependent Methods Flashcards
solation of bacteria and fungi into ___form is the best way to study and characterize
bacterial cultures.
pure
For bacteria, pure culture isolation is usually accomplished by
preading a dilution
containing microorganisms on a solid medium so that a single cell or a colony-forming unit
occupies a well-isolated portion of the agar surface.
Cultures consisting of single type
colonies are
pure cultures
among the
culture-dependent techniques used for this purpose
streak plating
spread plating
s, are present in almost all natural environments
fungi
colony types of nonfilamentous form
yeast
One of the best techniques in visualizing fungal structures as they develop is the
slide culture technique
Several reagents are added to the growth on a slide to
rehydrate tissues, provide contrast between parts and background and detect certain
biomolecules such as
starch
agar plate for fungi
potato dextrose agar plate
reagents for fungi stianing
lactophenol cotton blue
positive stain
methylene blue
negative stain
nigrosin
bacterial sample
s. marcescens
mold culture
a. niger
. Prepare sterile __ plates and draw four quadrants at the base to guide
streaking.
nutrient agar
- Incubate the plates in an ___ position at ___˚C for 48 hrs.
streak plating
inverted
37oC
48 hours
. Draw about ___ ml from a 24-hr broth culture using a pipette with sterile
blue tips, and deposit into a sterile NA plate for spread plate
0.1ml
Using a sterile ___, spread the inoculum evenly over the
surface of the agar and let stand for few minutes with the lid ajar to dry.
L-rod spreader
- Prepare a bacterial smear.
- Stain the smear with methylene blue for 1 min.
- Wash with water and blot dry. Focus under the OIO.
- The microorganisms should be colored dark blue to violet with a bright
background. - Answer item 3-4 in the worksheet.
what staining
positive staining
Using an inoculating loop, apply a small loopful of bacteria to one end of a
clean microscope slide.
2. Add 1 to 2 loopful of nigrosine dye to the bacteria and mix thoroughly.
3. Spread the mixture over the slide by laying over a second glass slide onto
the drop and then pulling slides away from each other. Air dry only.
4. Focus under the OIO.
whwat staining
negative staining
setup of slide culture technique
clean filter paper inside the petri dish, then a sterile
bent glass rod on top of the filter paper. Secure a clean slide and place it over
the bent glass rod. Autoclave right-side up.
Aseptically cut a block of what media (and what size) for slide culture
PDA (1cm^2)
Repeat steps 3-4 using Rhizopus as the inoculum. Incubate at __3C for 48 hrs
30
Place a drop of ____ ___ ___on a clean slide and lay over the
coverslip from the set up. Observe under OIO.
lactophenol cotton blue
view module 3 results
+1
Name a general-purpose medium used for isolation and cultivation of bacteria.
indicate the purpose/uses/functions of the medium and enumerate the
components.
Nutrient agar (NA) is generally used for streak, spread, pour, slant, etc. In
general, its purpose is to provide a solid surface for cultivation to create separate
colonies in either pure or mixed culture. In addition, this provides nutrients for
non-fastidious bacteria that can also be used in stocking cultures. The
components of NA are peptone, beef extract, sodium chloride, agar, and distilled
water (these components may differ according to the manufacturer).
- Compare/contrast principles of positive and negative staining. (3 pts.)
Positive staining is coloring microorganisms on a clear or bright background
using basic dyes such as crystal violet, methylene blue, and safranin. Direct
staining enables easier viewing of cellular structures and characteristics.
Additionally, heat fixation is required for this staining. On the other hand, negative
staining colors the background surrounding the cells using acidic dyes with a
negative charge like nigrosin and Indian ink creating a silhouette effect useful for
visualizing structures difficult to stain directly. In this staining, heat fixation is
avoided because it can damage cellular characteristics.
aspergillus
septate/aseptate
septate
asexual spore of aspergillus
conidia
phylum of aspergillus
ascomycota
is rhizopus septate/aseptate
aseptate
asexual spore of rhizopus
sporangia
phylum of rhizopus
zygomycota
. Give two other reagents used in microscopic examination of fungi. Explain their
purposes.
Other common reagents used in the microscopic examination of fungi are
Schiff’s Reagent and Congo Red
often used with periodic acid,
detects polysaccharides in fungal cell walls by reacting with aldehyde groups to
produce a vivid magenta color, making it especially useful for identifying fungi
in tissue samples during histopathological analysis.
schiff’s reagent
binds specifically to chitin and cellulose in fungal cell walls, allowing clear
visualization of yeasts and filamentous fungi under the microscope. It often
produces a bright orange fluorescence, making it a valuable tool for rapidly
detecting fungi in clinical specimens and culture preparations.
congo red
Name a general-purpose medium used for isolation and cultivation of fungi.
Indicate the purpose/uses/functions of the medium and enumerate the
components
SDA
is a widely used medium for isolating and
growing fungi. It serves a general purpose in fungal culture and inhibits bacterial
growth due to its high acid concentrations. The components of SDA are peptone,
dextrose, agar, and water with an acidic pH of around 5.6.
saboraud dextrose agar
SDA is composed of
peptone
dextrose
agar
water
pH of SDA
5.6
