1st SEM MICROBIOLOGY FINAL PRACS

EXERCISE 1A
GRAM STAINING
GRAM (+) BACILLI – ARRANGED SINGLY GRAM (-) BACILLI – ARANGED IN CLUSTERS

GRAM (-) COCCI – ARRANGED IN CLUSTERS

SIMPLE STAINING
COCCI ARRANGED IN PAIRS

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ACID FAST STAINING
(+) ACID FAST BACILLI

- Gram positive will appear PURPLE

-
- Gram negative will appear RED

-
- All BACILLI are GRAM NEGATIVE. EXCEPT
o CORYNEBACTERIUM,
o MYCOBACTERIUM,
o AEROBIC SPORE FORMERS (BACILLUS), AND
o ANEROBIC SPORE FORMERS (CLOSTRIDIUM)
- All COCCI are GRAM POSITIVE with the exception of
o NEISSERIA and VEILLONELLA
- GRAM STAIN IS USUALLY NOT APPLIED TO SPIROCHETES (ACID FAST)

- ACID FAST
- Ziehl-Neelsen or Kinyoun Method
- Acid-fast organisms resist decolorization by dilute acids and retain the original stain. They appear red against a
blue background.
- Non acid-fast organisms are decolorized by dilute acids and take up the counterstain – they appear blue like the
rest of the smear

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EXERCISE 1B
SPECIAL STRUCTURES
SPORE METACHROMATIC GRANULES

CAPSULE FLAGELLA

Capsule – HISS
Metachromatic granules – LAMB STAIN (leofflers alkaline methylene blue)
Flagella – LEIFSON
Spores – DORNER or WIRTZ & CONKLIN STAIN

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EXERCISE 2
INOCULATION TECHNIQUES
BROTH (isasawsaw at ipipihit lang yung wire loop)
SLANT (A) wireloop
BUTT (B) straight wire
BUTT SLANT (C)

INTERRUPTED OVERLAP MULTIPLE INTERRUPTED RADIAL STREAK

According to physical state According to their composition

- Solid media – substances mixed in a - Synthetic media – exact chemical composition of the
solidified agar or albumin matrix. ingridients is known (ringer’s solution, locke’s solution)
- Semi solid media – used for keeping - Non-synthetic media – the precise composition of some or all
stocks of bacteria for long periods of of the nutritive elements used is not definitely known (meat
time extract broth, vitamin agar)
- Liquid media – without adding gelatin - Living tissue media – one in which living tissue cells are
or agar present. For cultivating rickettsiae and viruses. (mainland
tissue culture medium, tissue – plasma roller tube medium,
embryonated egg)
According to their use Others
- Simple media – ordinary media which are not - For anaerobes – chopped cooked meat medium,
enriched and in which ordinary organism are easily thioglycollate medium
cultivated (broth or plain agar media) - Antimicrobial susceptibility testing – muller-hinton
- Specific media – special media which are used for agar
the cultivation of particular organism (leoffler Selective media
blood serum medium, cooked meat tubes, - Bordet-gengrou agar – pertussis
Lowenstein medium) - Thiosulfate citrate bile salts
- Differential media – used fr the differentiation and - Theyer martin – Neisseria
identification of microorganisms ( nutrient gelatin, - Lowenstein Jensen medium – m. tuberculosis
citrate agar, mcConkey agar) - For fungi – SDA, PDA, Chlamyydospore agar,
mycosel agar
- For general use, - brain heart infusion, nutrient
broth, plain nutrient agar, trypticase soy agar,
tripticase soy both

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EXERCISE 3 indigenous microorganisms of man

Normal flora: Resident flora of the GIT:

skin eyes ears – Achromobacter spp.,
Candida albicans, and other yeasts, Aeromonas spp,
Corynebacterium spp., Alcaligenes faecalis,
Neisseria spp., Bacteriodes spp,
Peptostreptotoccus, Candida albicans and other yeasts,
Propionibacterium acnes, Clostridium spp,
Staphylococcus aureus, Corynebacterium spp.,
S. epidermidis, and Enterobacteriace,
Viridans Streptococci Enterococcus,
FLavobacterium spp,
Fusobacterium spp,
Lactobacillus spp,
Mycoplasma spp.,
Peptostreptpococcus,
Pseudomonas aeruginosa,
Staphylococcus aureus,
Viridans streptococci,
Vibrio spp.
Larynx, Trachea, Bronci, Broncioles, Alveoli and the accessory nasal sinuses – usually sterile.
Esophagus and stomach are usually sterile, but can be contaminated by bacteria whenever food is ingested.
Liver and gall bladder usually free from microbial contamination.
Genitourinary tract, the resident organism are usually found in the external genitalia, the anterior urethra and the
vagina. The remaining structures of the genitourinary tract are without permanent microbiota.
Blood and spinal fluid of healthy individuals are usually sterile.
EXERCISE 4 ENVIRONMENTAL SAMPLING
Surface sampling – incubate 35 deg for 48hrs
Air sampling – incubate 35 deg for 24 hrs – more than 10 colonies per plate (Unsatisfactory) Less than 10 colonies per
plate (satisfactory)
EXERCISE 5 Inactivation of microorganisms
1. Heat methods
Autoclave – steam under pressure – 15lbs/sqinch for 15-20 mins – most efficient method
Streaming steam – the Arnold sterilizer – for media, intermittent or fractional sterilization in the Arnold sterilizer is the
best – 20-30mins at 100C repeated for 3 consecutive days. Spores not killed by one such exposure are taken care of by
exposure to such temp for 3 successive days.
2. Filtration
Applicable to materials such as sugars, sera, tissue extracts. Etc. which cannot be sterilized by heat or any other method.
Bacterial filters are usually made of unglazed porcelain, diatomaceous earth, asbestos, etc. of different sizes and grades
of porosity. – berkefeld, Pasteur – chamberland, mandlers, Millipore, and the seitz filters. – filters are facilitated by
vaccum or pressure pump.

UV RADIATION On BACTERIA – UVL disrupts H-bonds in microbial cell resulting to the formation of thymine dimers which
produce lethal frame shift mutations.

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EXERCISE 6 inactivation of microorganisms
D

Zephiran – 70% alcohol – iodine –

merthiolate – sodium hypochlorite

s
CHEMICAL AGENT 1 control sample 2 (1min dilute susp) 3 (1 min heavy susp) 4 (3min heavy susp)
before disinfection
Zephiran Heavy Moderate to light No growth No growth
Merthiolate Heavy Moderate to heavy Moderate No growth
70% alcohol Heavy Moderate to heavy Light No growth
Iodine/betadine Heavy No growth No growth No growth
Sodium Hypochlorite Heavy Moderate to heavy Moderate No growth
Disinfectant – chemical agents applied to inanimate object
Antiseptic – substance applied to the skin/living tissue for the purpose of eliminating or reducing the number of bacteria
present. Does not kill spores, cannot be used as disinfectant
General Mechanism of action
Zephiran – cationic agents Distortion of cell membrane and loss of membrane permeability leading to leakage of
nitrogen and phosphorous containing compounds
Merthiolate (heavy metals) Poison the enzyme activity forming mercaptids with the sh-groups of cysteine groups
70% alcohol (alcohols) Disorganize lipid structure by penetrating into hydrocarbon region, denatures cellular
proteins
Iodine/betadine (iodizing Inactivate enzymes by converting functional sh groups to s-s form
agents)
Sodium hypochlorite Disinfection is due to liberation of free chlorine
(chlorine)

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EXERCISE 7 ANTIMICROBIAL SUSCEPTIBILITY TESTING
Susceptibility testing is most often It is rarely indicated when it is known that It is considered wasteful or grossly
indicated when the ethiologic agent is the microorganism has not yet developed misleading to request susceptibility test
known to be capable of producing resistance against a particular antibiotic as when the infection is not clear or when
resistance to the commonly used in the case of pneumococcus, Neisseria the specimen contains a mixture of
antimicrobial agents such as species and most pathogenic streptococci. normal flora in which the microorganism
Staphylococcus aureus, enteric bacteria probably has no relationship to the
and pseudomonas aeruginosa. disease being treated
2 methods used in conducting susceptibility testing - Disc method and Test tube method.
Paper disc impregnated with known concentration of different antibiotics are placed on Mueller hinton medium where the
microorganism has been inoculated.
Factors which will create variation in results:
- Size of inoculum,
- Size of antibiotic molecule
- Length of incubation.
A large zone of inhibition does not necessarily indicate a more effective antibiotic since a large molecule will diffuse from the disc for
only a short distance while a small one can diffuse a greater distance.
Some antibiotics are heat labile and will deteriorate under prolonged incubation.
Disc method can be used to determine bacteriostasis only. And is applicable to fast growing aerobes and facultative
microorganisms. It cannot be applied to obligate anaerobe, slow growing aerobes and capnophiles.
Test tube serial dilution method gives a fairly precise determination of susceptibility to a measured amount of the test
antibiotic. It can be used to determine bacteriostatic as well as bactericidal concentration of the antibiotic.
Recommended for:
- Blood cultures,
- when pt fails to respond to apparently adequate therapy,
- for pt who develop relapse after undergoing antibiotic treatment
Staphylococcus aureus Zone size Interpretation
Penicillin g (p70) 40mm Sus
Sulfamethoxazole (sxt25) 23mm Sus
Chloramphenicol (c30) 27mm Sus
Escherichia coli

Sulfamethoxazole (sxt25) 11mm Intermediate

Chloramphenicol (c30) 5mm Resistant
Tetracycline (te30) 22mm Susceptible
Penicillin G (p70) 3mm Resistant

Pseudomonas aeruginoa

Tetracyclin (te30) 13mm Resistant

Chloramphenicol (c30) 70mm Resistant
Vancomycin (va30) 3mm Resistant
Penicillin G (p70) 3mm Resistant
Factors in choosing an antibiotic for treatment
- positive morphological culture result
- resistance patterns of microbial flora
- properties of the antibiotic ( route of admin,pharmacokinetics)
- patient health status and allergies
- bacterial factors: size of infection, bacteria involved
- host factors: site, allergies, route, heaptic function, pregnancy
- drug factors: activity site, bactericidal/bacteriostatic route, cost

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EXERCISE 8 GRAM (+) COCCI
Staphylococcus
S. aureus S. epidermidis S. saprophyticus
Pigment Golden yellow White White
Coagulase (+) (-) (-)
Hemolysis Alpha positive Gamma (non- Gamma (non-
hemocytic) hemocytic)
Novobiocin sensitivity Sensitive Sensitive Resistant
Mannitol fermentation Positive Negative Negative
Catalase (+) bubble (+) bubble (+)bubble
Gram (+) cocci in irregular grape like clusters

Nawala yung saprophyticus ko :\

meron ba kasama?

MSA Catalase test Coagulase test

(-) (+)

Positive kung may bubbles.

(+)clot formation (-) clear

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STREPTOCOCCI
Streptococci do not produce catalase, oxidase
negative.

` No bubbles (-)
Gram positive cocci in chains in varying lengths
Produced grayish, translucent to slightly opaque circular small
colonies
Usually grown in blood agar medium because of their hemolytic
properties which are useful in their identification.
3 groups:
- alpha – hemolytic streptococci produce incomplete lysis of
the red blood cells whith the formation of a green zone
around colonies
- beta – hemolytic streptococci produce complete lysis of
erythrocytes with release of haemoglobin resulting in a clear
clorless zone around streptococcus colonies
- gamma – streptococci produce no hemolytic activity or
discoloration around the colonie

Growth on 6.5% NaCl (+) turbid |Bacitracin test,gamma hemolytic strep|OPTOCHIN TEST S.pneumococcus

Group D streptococci are distinguished by their reaction on bile esculin medium. They can hydrolyze esculin to 6,7
dihydroxycoumarin which reacts with an iron salt in the medium to form dark brown or black components
Group D can grow in salt broth, while non-enterococcal group D such as streptococcus equinus and streptococcus bovis
do not.
Produce either alpha or beta hemolysis or can be also be non-hemolytic

Streptococcus pneumonia - encapsulated lanceolate organism typically arranged in pairs but could also appear singly or
in short chains
Colonies are alpha hemolytic, small, shiny, flattened and the centers may be depressed due to autolysis.

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EXERCISE 9 GRAM NEGATIVE COCCI – NEISSERIA
GRAM (-) COCCI IN CLUSTERS (COFFEE BEAN SHAPED)
CHOCOLATE AGAR (+)
THAYER MARTIN MEDIUM
Type of culture – selective medium
VANCOMYCIN – effective against gram (+) bacteria
COLISTIN – effective against gram (-) bacteria and used
as a polypeptide antibiotic
NYSTATIN – inhibits fungi
Neisseria gonorrhoea
(+)glucose, (-) maltose, (-) sucrose
Yung N. meningitides, (+) (+) (-) wla akong pic 
gwendie
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Biochemical test: Sugars fermentation (+) Yellow, (-) RED/ORANGE

DEXTROSE (lavender cotton plug)
LACTOSE (white cotton plug)
MANNITE (yellow cotton plug)

(+) Yellow (-) red

Biochemical test: TRIPLE SUGAR IRON AGAR (TSI) K=red, A=yellow, H2S = black?
A/A + GAS K/A K/K K/A+ H2S
Slant: Acid Slant: Alkaline Slant: Alkaline Slant: Alkaline
Butt: Acid + gas Butt: Acid Butt: No Change Butt: Acid, no Gas, H2S
(Klebsiella pneumonia) (Shigella dysenteriae) (Pseudomonas aeruginosa) (+) (Salmonella typhi)
(E.coli)

K/A + H2s + gas TSI

(Proteus mirabilis)
0.1% glucose, 1% lactose, 1%
sucrose.
Indicator – phenol red
acid formation – turns yellow
Black butt– may H2S d/t
ferrous sulfide,
Kapag nag split – gas
produced, aerogenic culture
A-yellow – acid
K- red – alKaline
K/A- glucose fermented
A/A-lactose/sucrose/glucose
fermented
K/K- none fermented

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Indole Methyl red test Vi (vogues- Citrate Urease
proskauer)
Confirm ERLICH’S RGT METHYL RED ALPHA-NAPTHOL BROMTHYMOL BLUE PHENOL RED
atory rgt 0.3% CREATINE IN
40%Koh
(+)RED RING (+) RED (+) WINE COLOR (+) BLUE COLOR (+) FUSCHIA PINK
(-) ABSENCE OF RED (-) YELLOW (-) CHOCOLATE (-)GREEN COLOR (-) SALMON PINK
RING *MAY VARY DEPENDING
BROWN ON BRAND OF MEDIA,

TSI I M V C U D L M
Klebsiella pneumonia A/A +gas - - + + + + + +
Gram (-), single, Coccibacilli, non - - ++(IMVC)
motile ++++(UDLM)
Encapsulated, non sporeformer,
Lactose fermenter
(red mcConkey)
Escherichia coli A/A+gas + + - - - + + +
Gram (-), single, bacillus, motile, ++ - -
encapsulated , w/o spore, - +++
Lactose fermenter (red
mcConkey)
Green metallic sheen
Shigella dysenteriae K/A - + - - - + - -
Gram (-), single, bacillus, -+--
non motile, no capsule/spore, -+--
non lactose fermenter Yellow
mcConkey)
Salmonella typhi K/A + sm amt - + - - - + - +
of H2S
Gram (-), single, bacillus, motile,
-+--
encapsulated, no Spore, -+-+
non-lactose fermenter (yellow
mcConkey)
Proteus mirabilis K/A + H2s + - + - + + + - -
Gram (-), single, bacillus, motile, gas
no capsule/spore, non lactose -+-+
fermenter (yellow mcConkey) ++ - -
swarming phenomenon
Pseudomonas aeruginosa K/K Seller’s - - - + - - - -
g/g
Gram (-), Cluster, bacillus, green/green
motile, encapsulated, no ---+
spore, non lactose fermenter ----
(yellow mcConkey)

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McConkey medium

Lactose fermenter Non-Lactose fermenter

Enterobactericae

TSI

A/A+ gas K/A + gas K/A + gas + H2S K/K

Lactose fermenters Non lactose fermenters Non fermenters
E.coli Shigella
Salmonella
P. aeruginosa
K.pneumonia p.nettgeri Proteus vulgaris Acitenobacter calcoaceticus
E.aerogenes M.morgagnii P. mirabillis
serratia

Seller’s test

IMVC test Urease Urease

G/G Pseudomonas
Aeruginosa
++-- + B/G acinetobacter calcoaceticus
P.rettgeri +
E.coli B/B Alkaligenes fecalis
M.morgagnii
proteus

--++ -
K.pneumoniae All shigella
-
E. cloacae CONFIRM BY
Salmonella
SEROTYPING
CONFIRM BY
SEROTYPING
Urease Citrate Indole

+ - +
K.pneumonia P.rettgeri p. vulgaris
e
- + -
E. aerogenes m. morgagnii P.mirabilis

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Lowenstein-jensen medium- Cultivating medium for Loeffler’s coagulated serum slant – medium used in
mycobacterium tuberculosis cultivating corynebacterium diptheriae

Presence of Metachromatic Granules (LAMB Stain)

•Loeffler’s Coagulated Serum Slant )C. diptheriae)
•+ Elek Test (presence of precipitin lines)

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Anaerobic Chopped cooked meat medium –
Gaspak jar - sealed with petrolatum to prevent entrance of
atmospheric oxygen
- Pieces of meat provide low redox potential

Thioglycollate medium
Sodium thioglycollate – the reducing substance that lowers
the redox potential making it ideal for anaerobic culture
Resazurin – indicator of redox potential, narrow pink layer

Medyo yung upper

part netong medium na
to,

Aerobic B. subtilis with spore (yung nasa gitna ng pic)

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Pseudomonas

*Pyocyanin (pigment produced yung sa taas) – can color the

pus in a wound grows as non-lactose fermenting (colorless) colonies on
Pyoverdin (fluorescein) – yellow green pigment that McConkey or EBM agar.
fluoresces under UVL
sa pseudomonas, may Metallic sheen on TSI agar
coupled with blue green pigment on ordinary nutrient
agar, grape like aroma.

Meron din sa E.coli na green metallic sheen sa EMB

AGAR.
Yung nasa picture, E.coli.

*Galing sa micro lab manual yung ibang notes dito.

*Yung ibang pictures from old trans and google,
*kung may kulang, sana hindi lumabas o itanong yung
kulang.
*kung di mo naaral yung kulang dito, idasal mo nalang
na yung nandito lang yung lalabas.
*sana pumasa tayo lahat dito.
*salamat

~Hi ke ayie, Ckaye at ke regis. Si dysphoric may-lyn.

And sa mga nakakasama ko mag aral sa labas.
~kim! yung Tumbler ko balik mo na! haha
~salamat sa mga gumagawa ng trans. Sana hindi kayo
tamarin. More power to you guys.
lol
-donRM

“That in all things, God may be glorified”

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