1. Submitted By:
Daisy Saini
B.Sc. Microbiology
Final Year
Branch : B.Sc. Microbiology
Submitted To:
Dr. Seema Bhadauria
(Head, Department of
Microbiology)
Jaipur
A Project report submitted to
JECRC University
IDENTIFICATION AND ISOLATION OF NON-LACTOSE FERMENTING
BACTERIA FROM VARIOUS CLINICAL SAMPLES.
TOPIC:
Under the guidance of Dr. Saroj Hooja
(Professor ,Department of microbiology) S.M.S.
Medical hospital,Jaipur

2. CONTENT
S.No. Title
1. ABSTRACTS
1. INTRODUCTION
2. AIMS AND OBJECTIVES
3. MATERIALS AND METHODS
4. OBSERVATION
5. RESULT
6. SUMARRY

3. ABSTRACT
Non-lactose fermenting gram-negative bacilli have emerged as
important healthcare-associated pathogens. This study was
undertaken to identify the non-lactose fermenters isolated from
various clinical samples by various methods to assess their
clinical significance. The Non-Lactose fermenters were
identified using standard protocols.

4. INTRODUCTION
Non lactose Fermenting gram-negative bacilli(NLFGNB)
are a group of aerobic, non-sporing,
bacilli/coccobacilli that are either do not utilize glucose as a source of
energy or utilize it oxidatively.They occur as saprophytes in the
environment and also found as commensals in the human gut.
This group includes numerous organisms but the ones
which are known to cause infections are Pseudomonas
aeruginosa , Acinetobacter baumannii , Proteus vulgaris ,
Proteus mirabilis and Salmonella typhi and members of genus
Citrobacter etc.

5. NLF Bacteria have emerged as important healthcare-associated
pathogens. They have been incriminated in infections, such as,
septicemia, meningitis, pneumonia, urinary tract infections (UTI), and
surgical site infections (SSI). NLF Bacteria are innately resistant to
many antibiotics.
The NLF Bacteria were identified using a standard protocol that
included tests for motility, oxidase production, catalse test , TSI(triple
sugar iron test), Indole production test, Simmon’s citrate test , urease
test , Phenylalanine deaminase production test and by growth on
different agar media.

6. AIMS AND OBJECTIVES
To isolation and identification Non-lactose fermenting (NLF)
bacteria in various clinical samples.
Distribution of Non-lactose fermentative bacterial infection
according to different clinical samples, wards, sex and age group.
Distribution of NLF bacterial infection in different clinical samples,
wards, sex and age group according to their profile.

7. MATERIALS AND METHOD
Samples were collected from urine, pus/wound, blood, ascitic/plural
fluids and were analyzed for colonial morphology and routine biochemical
identification.
Specimen
Blood agar
MacConkey
agar
Growth
Lactose
Lactose
fermenter
Non -Lactose
fermenter
No Growth
Mac-Conkey Agar is inoculated with tested organism using streak or
spread plate technique then incubated at 37˚C for 24-48 hours and
observed.

8. Pink colony
appears = LF
Bacterial
colony
Pale or colorless
colony growth =
NLF Bacterial
colony
MacConkey agar is a selective and differential(The differential ingredient
is lactose) culture media commonly used for the isolation of enteric Gram-
negative bacteria. Because:
Bile Salts: selective agents and inhibit Gram positive organisms.
Crystal Violet: Generally inhibited Gram positive bacteria.
Neutral Red: pH indicator. which is red in color at pH’s below 6.8.
When lactose is fermented, the pH of the medium decreases and this
changing the color of neutral red to pink .

9. Identified NLF colony further goes threw Biochemical tests for their
identification according to their profile.
Biochemical tests are the tests used for the identification of bacteria species
based on the differences in the biochemical activities of different bacteria.
Biochemical tests are the quickest and easiest tests for identifying bacteria
because :-
Microorganism’s visual identification is not always possible because
structural differences, which may differentiate one species of bacteria from the
other, are not discernible sometimes even under a microscope.
Genetic testing is expensive and time consuming.
Each species of bacteria has a well-defined set of metabolic activities
different from all other species. These biochemical fingerprints are properties
controlled by the bacterial enzymes.

10. Biochemical Characterizations
•Simmon’s Citrate Utilization Test- Use
to identify if the organism can use citrate as its sole
carbon source or not by the use of enzyme citrase.
Citrate positive medium will be turn into intense
Prussian blue color.
Principle-
Hydrolyzation of citrate by citrase enzyme
• Oxaloacetic acid and acetic acid---Hydrolyze---Pyruvic
acid and CO2
• CO2 react with compoments in medium and produced
Na2Co3(Alkaline compound)
pH of medium changes
• bromothymol blue indicator, turn media from green to
blue.

11. •Urease Test- Use to identify if the organism
capable of hydrolyzing urea using urease or not
Urease positive medium will be turn into pink color.
•Principle-
Hydrolysis of urea by
urease
• release of ammonia(Weak base)
and carbon dioxide.
pH of media changes
• turning the indicator phenol red
from its original yellow color to
bright pink.

12. •Triple Sugar iron test(TSI)-
A test which has three sugar Lactose,
Sucrose and Glucose in the concentration of
10:10:1. It also contain Iron(Ferrous sulfate)
which is an indicator of H2S formation and
Phenol red which is Indicator of acidification
(It is yellow in acidic condition and red under
alkaline conditions).It also
contains Peptone which acts as source of
nitrogen. Some organisms generate gases,
which produces bubbles/cracks on the
medium.TSI is a semi solid media
having slant and butt.
This used to identify organism’s ability to-
• Ferment sugars
• Produce hydrogen sulphide.

13. If lactose (or sucrose) is fermented, a large amount of acid is
produced, which turns the phenol red indicator yellow, both in
butt and in the slant(A/A)
If the small amount of glucose is fermented, the butt will be
yellow ( because butt comparatively have more glucose
compared to slant i.e. more media more glucose).(K/A)
If neither lactose/sucrose nor glucose is fermented,
both the butt and the slant will be red (K/K).
If H2S is produced, the black color of ferrous sulfide is
seen
•Interpretation –

14. •Sulphide Indole Motility(SIM)-
Use to Test the ability of organism to do-
1- Reduce Sulphur-Blackening of the media
indicates hydrogen sulfide production.
2-Produce Indole- After addition of 5-10
drops Kovács reagent, a pink ring at the top of
the tube indicates a positive indole result.
3-Motility- Growth feathering away from the
stab line creating a cloudy appearance in the
media indicates motility.
Principle of indole test-
• Metabolization of tryptophan by tryptophanase
• The formation of pyruvic acid, indole and ammonia
• The presence of indole is detected by addition of Kovac’s reagent(Rosindole dye
formation).

15. •Phenylalanine Deaminase
Production- Use to test the ability of
organism to produce the enzyme
deaminase.
After adding a few drops of ferric chloride in
test medium a green colored compound
turn the top of the medium dark green thus
indicate positive result.
Principle-
(PPA)Amino acid
Ferric chloride act as chelating agent and combined with
PPA and gives dark green color.

16. Use to identify the presence of catalase
enzyme by using hydrogen peroxide. The
enzyme catalase mediates the breakdown of
hydrogen peroxide into oxygen and water
Immediate bubble formation in given by
catalase positive isolate.
Catalase test -
Oxidase test –
The oxidase test is used to identify bacteria
that produce cytochrome c oxidase, an
enzyme of the bacterial electron transport
chain. When present, the cytochrome c
oxidase oxidizes the reagent tetramethyl-p-
phenylenediamine(TMPD) to indophenols
thus indicated by the development of a dark
purple colour

17. OBSERVATION
Name of
organism
Pseudomonas
aeruginosa
Acinetobacter
baumannii
Proteus
mirabilis
Proteus
vulgaris
characteristics
Gram staining Negative Negative Negative Negative
Motility test Positive Negative Positive Positive
Catalase test Positive Positive Positive Positive
Oxidase test Positive Negative Negative Negative
Indole test Negative Negative Negative Positive
Citrate test Positive Positive Positive Positive
Urease test Negative Negative Positive Positive
H2S
production
Negative Negative Positive Positive
Phenylalanine
deaminase
test
Negative Negative Positive Positive

18. OBSERVATION
Proteus Mirabilis Proteus Vulgaris

19. Pseudomonas Acinobacter

20. In During 4 month period only 381 isolates were
identified as non-lactose fermentative bacteria at
the Bacteriology section of Department of
Microbiology and Immunology, SMS Medical
College, Jaipur .Then further their distribution
according to patient’s age, sex, ward and clinical
samples was found out.
RESULT

21. Medical
ICU
Surgical
ICU
Surgical
ward
Burn ward
Total no. 381 381 381 381
NLF Bacteria 102 96 110 73
26.77% 25.19% 28.87% 19.16%
Figure - 1
Figure-1 (Distribution of NLF Bacteria according to Wards/ICUs)
shows that, maximum distribution of Non-lactose fermenters was found
in surgical ward (28.87%) while in burn ward it was slightly lower
(19.16%).Surgical ICU(25.19%) and Medical ICU(26.77%) shows similar
distribution.

22. 31.49%
28.34%
27.29%
6.29%
6.56%
Repiratory specimens
Pus/Wound swab
Urine
Blood
Others*
Repiratory
specimens
Pus/Wound
swab
Urine Blood Others*
NLF Bacteria 120 108 104 24 25
Total no. 381 381 381 381 381
Figure-2
Figure – 2 (Distribution of Non-lactose fermentative bacterial
infection in different clinical samples)
shows that highest culture positivity was seen in respiratory samples
(31.49%), while blood and others samples collected have shown lower
growth only 6.29% and 6.56% samples were culture positive. Pus/Wound
swab(28.34%) and Urine(27.29%) shows similar distribution.
*Various samples as tissue, chest drain, ET tip, body fluids etc. were included together in other samples.

23. 15-29 30-44 45-59 >60--
Total no. isolate 171 185 223 357
NLF Bacteria 57 82 101 141
14.96% 21.52%
26.50%
37%
Figure - 3
Figure-3 (Correlation of NLF Bacteria with age of patients) shows that
maximum number of non-lactose fermenters were isolated in the age
group >60 years (37%) while only 14.96% were isolated in the age group
15-29 years. the number of NLF bacteria distribution was increasing with
age of patients.

24. 66.6%
33.3%
Figure-4
Male
Female
Figure-4 (Correlation of NLF Bacteria
with sex of patients)
shows that distribution of non-lactose
fermenter in male and female patients.
The male to female ratio was found to be
of 2: 1.
Pseudomonas
species
Acinetobacter
baumanni
Proteus species
55.64%
29.65%
14.64%
Figure-5
Total No. of isolates
Figure-5(Profile of non-lactose
fermenting gram negative
bacilli)
shows that highest culture
positivity was seen in
Pseudomonas species(n=212)
55.64%, while only 14.69% (n=56)
non-lactose fermenters belonged
to Proteus species and 2nd most
common NLF found was
Acinetobacter baumannii (n=113)
29.65%.

25. Pseudomonas
species
Acinetobacter
baumannii
Proteus
mirabilis
Proteus vulgaris
Burn ward 47 18 6 2
Surgical ward 61 29 14 6
Surgical ICU 52 32 8 4
Medical ICU 52 34 12 4
52 34 12 4
52
32 8 4
61
29 14 6
47 18 6 2
Figure - 6
Figure-6 (Distribution of NLF Bacteria in selected wards and ICUs )shows
that
The highest distribution of Pseudomonas species was found in Surgical ward (n=61,
28.77%).
Highest distribution of Acinetobacter baumanni was found in Medical ICU (n=34 ,
30.08%).
Highest distribution of Proteus mirabilis was found in Surgical ward (n=14 , 35%).
Highest distribution of Proteus vulgaris was found in Surgical ward (n=6 , 37.5%).

26. Pseudomon
as species
Acinetobact
er
baumannii
Proteus
mirabilis
Proteus
vulgaris
Respiratory specimenPus 77 38 5 0
Pus 64 29 9 6
Urine 55 24 14 11
Blood 7 10 7 0
Others 9 12 3 1
Figure - 7
Figure-7 (Distribution of Non-Lactose fermenters in various clinical
samples) shows that majority of NLF isolates were isolated from respiratory
specimens 120(31.49%). Among these Pseudomonas species (64.16%) was the most
common isolate. Least NLF were isolated from blood 24(6.29%). In blood samples the
most common isolate was Acinetobacter baumnnii (41.66%) and the maximum no. of
Proteus species was found in urine samples in which Proteus mirabilis is 13.46% and
Proteus vulgaris is 10.57%.

27. Pseudomona
s species
Acinetobacte
r baumannii
Proteus
mirabilis
Proteus
vulgaris
Male 138 76 29 13
Female 74 37 11 3
53.09%
29.68%
11.41%
5.11%
59.29%
29.6%
8.66% 2.36%
Figure - 8
Figure-8 (Distribution of Non-Lactose fermenters according to sex)shows
the distribution of NLF bacteria were higher in male patients n=254 (66.66%) then
female patients n=121 (33.33%).
In overall samples organisms Pseudomonas species was predominent one in male it
shows 53.09% distribution and in females 59.2% cases was reported. Other organisms
also shows higher rate of infection in male patients.

28. SUMMARY
Identification and isolation of NLF Bacteria include some protocols
that include test for motility, oxidase production, TSI(triple sugar
iron test),Indole production test, citrate test, urease test,
Phenylalanine deminase production test and by growth on
different agar media.
There are aprox 20 types of NLF Bacteria commonly found in
hospital environment and infect human and the most common
species are Pseudomonas aeruginosa, Acinetobacter baumanii,
Proteus vulgaris, Proteus mirabilis. In which the most abundant
organism is Peudomonas species.