Independent Investigation of • (1) Gram staining which group

Unknown Bacteria
Important bacteria according to their cell wall
composition (gram positive and gram

• Maidy Andrea B. Mejorada, RMT

methods in negative), morphology and
arrangement, this procedure will
Identifying allow microbiologists to decide which
further steps are needed
unknown • (2) Use of appropriate culture media
bacteria (selective, differential and special)
provide an easily isolation of
intended organism and lastly

1 2

Schematic pattern on Bacterial Identification

• (3) the use of important biochemical
Important test facilitate a reliable microbial
identification. One must bear in mind
methods in that other method and procedure
must be considered such a serological
Identifying test, animal inoculation, phage
typing, amino acid sequencing,
unknown protein analysis and DNA base
composition are other advanced
bacteria methods for a successful microbial
identification

3 4

Blood & Urine INTRODUCTION

Blood for culture is probably the most important single
specimen submitted to the microbiology laboratory for

Culture, Water Blood Culture

examination. It helps provide a clinical diagnosis as well as
specific etiologic diagnosis.
The presence of microorganisms in the patient’s blood

Analysis
reflects active and possibly spreading infection in the tissues.
The prognosis of such bacteremia or septicemia may depend
on its prompt recognition by bacteriologic means.
Subsequent initiation of specific therapy based on laboratory
MAIDY ANDREA B. MEJORADA , RMT findings may prove to be lifesaving. Likewise, a negative
culture would prove helpful in ruling out microbial etiology.

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1
 11/12/2022

PROCEDURE

1. Blood specimen (sterile)

1 2 3 4 5 6
MATERIALS 2. BAP, Chocolate agar and Thioglycolate broth
3. Candle jar
Extract 10 mL Transfer 5 mL Note: The Incubate 37 Streak the BAP and Perform
blood to a thioglycolate chocolate agar,
blood thioglycolate broth is boiled
degrees incubate the BAP at biochemical
aseptically. Centigrade 37 degrees testing for
broth and the for 10 minutes Centigrade and the
other 5 mL to a before use to for 24 hours. chocolate agar in a microbial
bottle with BHI lessen the candle jar at 37 identification
agar and BHI oxygen content. degrees Centigrade.
broth.

7 8

Rationale
Urinary tract infections (UTI) are one of the most commonly
encountered acute infectious diseases. Most UTIs occur as a
result of bacteria ascending the urethra and entering the
urinary bladder.
Urine specimens for culture are collected when the following
syndromes are suspected:
Urine Culture cystitis, pyelonephritis, asymptomatic bacteriuria, and less
commonly acute prostatitis, pyelonephric abscess, and
urosepsis.
Among the bacteria most commonly isolated from patients
with acute uncomplicated cystitis are Escherichia coli,
Klebsiella species, and other Enterobacteriaceae and
Staphylococcus saprophyticus.
Group B Streptococcus are markers of colonization in
pregnant women.

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Materials
Compound Microscope
Urine cultures are evaluated with consideration
given to factors such as colony count, type and Sterile wide-mouthed container
Principle number of organisms growing, type of MacConkey agar plate
specimen and the clinical condition of the
patient. Blood Agar plate
Calibrated inoculating loop
Alcohol lamp
Midstream catch urine specimen

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2
 Wash and collect a morning midstream catch urine a sterile container.
OBJECTIVES
Wipe the outside of the container with disinfectant and close it tightly.

After collection, bring to the laboratory and should be processed within 1 hour after
collection. To analyze water sample for the detection of
If it cannot be processed immediately, urine should be kept in the refrigerator at 4OC coliform organisms,
and must be processed not longer than 18 hours.

Procedure Transfer approx. 1.0 mL of urine sample in a small sterile test tube. Hold the tubes to
To estimate the number of organisms in water
the light and examine urine for color, turbidity and pH. Note the odor of the urine.
Using the calibrated loop, get a loopful of the urine sample (from the original
Water Analysis sample,
container) and streak onto the BAP.
Repeat step #5 and streak onto the MacConkey agar plate.
To know the different water borne diseases and
Incubate all plates at 35OC to 37OC for 24 hours. the causative agents, and
Examine the plates for any growth.
To be aware of the present condition of the
Count the colonies on BAP and report the number of organisms per milliliter. environment with emphasis on water pollution.

13 14

INTRODUCTION
Water provides a habitat for many different microorganisms.
Among bacteria most commonly found in natural waters are
The importance of water contamination in the spread of infections is so sulfur bacteria, iron bacteria, free living spiral forms, and
well recognized and controlled in most countries by public health measures that spore-formers. Since water is in contact with the soil, many
it is difficult for most of us to realize how serious water contamination can be. common soil inhabitants, such as Bacillus subtilis are usually
Unfortunately, epidemics of water borne diseases such as cholera, dysentery, present. However, the important facts to remember about
and typhoid fever still occur in parts of the world today and these diseases could the flora of water are the pathogenic microbes. These
easily become epidemics if not rigidly controlled.
microorganisms should not be present in drinking water for
human consumption. Hence, water that contain pathogenic
organism is regarded as fecally contaminated.

15 16

a. Water samples from different sources f. Sterile pipettes

A test for fecal contamination is accomplished by looking for (1 & 10 ml)
an organism which occurs only in feces and never as free-
living in nature. There are several microbes which are used
as indicator for fecal contamination which
METHODS AND b. Inoculating loop g. Alcohol lamp

include, Escherichia coli, Streptococcus faecalis, Clostridium

species and Candida albicans. The findings of these microbes
MATERIALS c. Fermentation tubes h. Sterile water
d. Plated EMB i. Nutrient agar
are sufficient evidences that the water is not safe for
drinking purposes and therefore suggest fecal e. Colony counter
contamination.

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3
 1st Day-
PROCEDURES Presumptive Test
1st Day- Colony Counting Pipet 1 ml of water sample to test
a tube with inverted fermentation
tube containing 9ml Lactose broth.
1. Prepare serial dilution of the water sample, 1:10, 1:100,
up to 1:1,000,000 using sterile distilled water. Incubate at 37◦C for 24 hrs.
2. Pipet 1 ml of each dilution in a sterile plate starting from Observe tubes for gas production.
1:1,000,000 or to 1:10.
A positive presumptive test is one
3. Pour about 15 ml of the agar into the plate with diluted with gas formation.
water. Mix by swirling and allow to solidify.
If tube is without gas, stop the
4. Incubate at 37◦C for 24 hrs.
analysis. If there is gas production,
5. Count the number of colony and report it as colony proceed to confirmatory test.
forming units/ml(CFU/ml)

19 20

1. Streak EMB plate with organism from the test tube with
gas production.
2. Incubate at 37◦C.
3. After incubation observe for:
2nd Day- Escherichia coli -
Confirmatory Enterobacter species -

Test Enterobacter aerogenes

1. A positive confirmatory test constitutes a growth of
typical colonies.
2. Proceed to completed test.

21 22

3rd Day- Completed 4th Day- Reading of

Test Biochemical Test
1. Inoculate the distinct colonies to a fermentation tube After 24 hour incubation at 40◦C, read the
with lactose broth. biochemical test by adding appropriate reagents
2. Incubate at 37◦C. to the different culture media.
3. Make a gram stain and observe for the presence of
gram negative, non-spore forming bacilli.
4. After incubation, observe for a gas formation in
lactose broth. A positive completed test is the
production of gas in lactose broth and a gram-negative
non-spore forming bacilli.
5. To differentiate the fecal from the non-fecal
contaminations, inoculate the isolated bacteria to
SIM, MR-VP, Simon citrate for the IMViC test.

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4
 Principles of Identification

• Commercial identification systems fall into one of five

categories or a combination thereof:

Automation in • (1)

Microbiology Manual Multitest

• (2)

• (3)
Laboratory Systems • (4)

• (5)

• can be facilitated by the use of automated or

M A I D Y A N D R E A B . M E J O R A D A ,
R M T packaged kit systems by which organisms are
identified with computer-assisted databases or
computer-derived numeric codes.

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Analytical Profile Index

Accuracy for commonly isolated
Enterobacteriaceae, such as Escherichia coli,
• The system for identification of gram-negative API 20E. Klebsiella pneumoniae, and Proteus mirabilis, has
been reported to be 87.7% at 24 hours of
• This system has a series of 20 cupules attached to a plastic strip. incubation.
• Inside the cupules are lyophilized, pH-based substrates.

• A bacterial suspension made in saline is used to rehydrate the reagents in the For less commonly isolated Enterobacteriaceae, the
accuracy is 78.7% at 24 hours. bioMérieux markets
cupules.
many multitest API systems, including systems for
• The principles of the tests are the same or similar to the principles of tests gram-positive cocci, nonfermentive gram-negative
bacilli (20NE), and a rapid system for identification
performed in test tubes. of the Enterobacteriaceae in 4 hours (RapID 20E).
• Some of the cupules, such as those for amino acid deaminases and dehydrolase,
require a mineral oil overlay.

• The strip is incubated 18 to 24 hours at 35° C,

27 28

Rapid and Automated

Identification Systems The MicroScan System (Beckman Coulter, Brea,
CA) consists of plastic standard-sized, 96-well
microtiter trays in which 32 reagent substrates are
included for the identification of bacteria and yeasts.
Two systems are available: __________ and
MicroScan ___________________

. The clinical outcome of Systems

rapid and accurate (2) subsequent selection Some trays, called combo trays, include broth
reporting of results (1) early diagnosis of appropriate microdilutions of various antimicrobial agents for
should directly affect antimicrobial therapy. performing susceptibility tests along with
patient care in two ways: biochemical tests for identification

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5
 • Each biochemical reaction is repeated three
TREK Diagnostic System
times in the 96-well plates; each plate is

• The TREK Diagnostic System (ThermoFisher Scientific) offers two automated designed to test three separate organisms.
All tests are read on the Sensititre
systems: ____________________ and ____________________
Autoreader for the presence or absence of
• The Sensititre system uses ________________________ fluorescence. Results are available as soon
as 5 hours, although incubation can be
• The system comprises 32 biochemical tests, including selected classic
extended to overnight if needed. The results
biochemical media reformulated to yield a fluorescent signal. are transmitted to a computer for analysis

• The biochemical test medium, along with an appropriate fluorescent indicator, and identification.

is dried into the individual wells of the Sensititre plate.

31 32

• The card is then placed in the reader-incubator

module of the instrument, where it is optically
scanned and read periodically. The computer
software collates the readings and matches them to
the automated database for final identification.

• The reader/ incubator can accommodate 15, 30, 60,

Vitek 2 System
or 120 cards depending on the system.

• Numerous cards are available for gram-positive

cocci, coryneforms, fermentative gram-negative
TREK Diagnostic System bacilli, nonfermentative gram-negative bacilli,
Haemophilus, Neisseria, and yeasts

33 34

BD Phoenix Automated Microbiology System

• The BD Phoenix Automated Microbiology System (BD Diagnostic

• Systems) was released in the United States in 2004.

• Once the 136-well combination panels are inoculated, it is a totally hands-off system that
can hold 100 panels, 99 samples and 1 control.

• Panels are read every 20 minutes for up to 16 hours.

• Results are generally available in 2 to 12 hours for bacteria and 4 to 15 hours for yeasts.
Vitek 2 System
• Various colorimetric and fluorometric indicators are used for identification, and a
colorimetric redox indicator is used for antimicrobial susceptibility testing.

35 36

6
 • The fully automated Biolog OmniLog ID System is
based on the organism’s ability to utilize 95 carbon
sources.

Biolog OmniLog ID • One 96-well plate is used for each organism. The

System reduction of tetrazolium violet is used as the indicator

system.

• The Biolog database is one of the largest, with more

than 2500 species or taxa.

BD Phoenix Automated Microbiology System • Panels are available for aerobic and anaerobic gram-
positive and gram negative bacteria and yeasts.

37 38

identification is based on the 9- to 20-carbon fatty acid composition of

microorganisms

Sherlock
Microbial The Sherlock system examines which fatty acids are present as well as their
relative concentration (percentage). Fatty acids are located in the plasma
membrane of bacteria and, depending on environmental conditions, are
Identification modified by the bacteria.
For this reason, it is important that growth conditions are well standardized
System for accurate identification.

The standard incubation conditions for most aerobic bacteria are tryptic soy
Biolog OmniLog ID agar incubated at 28° C for 24 hours

System

39 40

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