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Methods of isolation and identification pathogenic and potential pathogenic bacteria from skins and tannery effluents.

Authors:
Anne Lama at The University of Northampton, UK
Anne Lama
  • The University of Northampton, UK
Margaret Bates at The University of Northampton
Margaret Bates
Anthony Dale Covington at The University of Northampton
Anthony Dale Covington
Paula Antunes at The University of Northampton
Paula Antunes
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Abstract and Figures

Currently there is no standard protocol available within the leather industry to isolate and identify pathogenic bacteria from hides, skins or tannery effluent. This study was therefore carried out to identify simple but effective methods for isolation and identification of bacterial pathogens from the effluent and skins during leather processing. Identification methods based on both phenotypic and genotypic characteristics were investigated. Bacillus cereus and Pseudomonas aeruginosa were used as indicator bacteria to evaluate the isolation and identification methods. Decontaminated calfskins were inoculated with a pure culture of the above mentioned bacterial species followed by pretanning and chromium tanning processes. Effluent samples were collected and skins were swabbed at the end of each processing stage. Bacterial identification was carried out based on the phenotypic characteristics; such as colony appearance on selective solid media, cell morphology following a standard Gram-staining and spore staining techniques, and biochemical reactions, e.g., the ability of a bacterial species to ferment particular sugars and ability to produce certain enzymes. Additionally, an identification system based on bacterial phenotypic characteristics, known as Biolog® system was applied. A pulsed-filed gel electrophoresis (PFGE) method for bacterial DNA fingerprinting was also evaluated and used for the identification of the inoculated bacteria. The methods described in the study were found to be effective for the identification of pathogenic bacteria from skins and effluent.
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JALCA, VOL. 108, 2013
48


methoDS of ISolatIoN aND IDeNtIfICatIoN of PathoGeNIC
aND PoteNtIal PathoGeNIC baCterIa from SkINS aND
taNNery efflueNtS
by
LaMa, M. BateS, a. D. Covington, S. C. aLLen anD a.p.M. antuneS*
Institute for Creative Leather Technologies, The University of Northampton
Northampton, UK.
Centre for Sustainable Waste Management, The University of Northampton
Northampton, UK.
Sports, Exercise and Life Science, The University of Northampton
Northampton, UK.
AbstRAct

 

            
  
 
      
    Ba cillus cereus 
Pseudomonas aeruginosa
         



   
              
        
      
      
  

          

       
       
         
      
        

Resumen
             


 
  


         
Bacilos céreos  Pseudomonas aeruginosa 
             

       




              
      
    

   

    

    





JALCA, VOL. 108, 2013
iSoLation anD iDentiFiCation oF BaCteria 49
IntRoductIon
            
        


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
        
BacilluscereusEscherichia
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aeruginosa3 

      
      
     
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     
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     
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    
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     
         
        
 
         
       

mAteRIAls And methods
Calfskins (Latco Ltd., UK) were used as raw materials.
Soaked, f leshed and salted calfskins were cut into small
pieces weighing approximately 100g each. The skin pieces
were washed w ith au toclaved sterile t a p w ater and
decontaminated using sodium hypochlorite (NaOCl, 125 mg/
ml). The skins were treated with NaOCl three times, for 3
minutes each time at room temperature. The decontaminated
samples were washed thoroughly with sterile water and the
flesh side of the skin pieces were inoculated with pure
cult ures of Bacillus cereus ATCC11778 (Oxoid, UK) and
Pseudomonas aeruginosa ATCC10145 (Oxoid, UK). The
number of inoculated B. cereus and P. aeruginosa on each of
the calfskin piece was approximately 108 colony forming
units (cfu) and 1010 cfu respectively. Bacterial species were
inoculated on the flesh side, as the epidermis layer (the outer
side of the skin) may prevent penetration of the bacterial
species through the cross-section of skins. Pure cultures of B.
cereus ATCC11778 and P. aeruginosa ATCC10145 will be
referred to as controls in this study. The bacterial inoculums
were collected from the mid-exponential growth phase as the
physical and chemical properties of the bacterial cells are
most consistent during this phase.12-13 The time required to
reach the mid-exponential phase was determined by plotting
growth curves for each of the bacterial species over time prior
testing (see Figure 1 as an example of a growth curve).




         

     

               

     

     

      








                  


JALCA, VOL. 108, 2013
50 iSoLation anD iDentiFiCation oF BaCteria
  
g

     
              
             


       
      
      

               

    









               




   
B. cereus
P. aeruginosa
   



  

B. cereus P.
aeruginosa




        



           
B. cereus



P. aeruginosa
PseudomonasP.aeruginosa




        





    


B. cereus
P. aeruginosa











B. cereus




 




P. aeruginosa
Pseudomonas



  

JALCA, VOL. 108, 2013


     




             

               

centrifuged cells were suspended in 0.15 ml lysis buffer (6
mM tris-HCl (pH 7.6), 1 M LiCl, 100 mM EDTA, 0.2% (w/v)
deoxycholate, 0.5% (w/v) sodium lauryl sarcosine). A 0.7%
(w/v) low melting point agarose (Bio-Rad, USA) was prepared
in PET-IV buffer and dissolved by using a microwave. The
melted or dissolved low melting-point agarose was stored at
4°C and re-melted as required. A bacterial suspension, in
lysis buffer, was agitated with 0.15 ml of the prepared molten
agarose (approximately 50°C), dispensed in a plug mould
(Bio-Rad, UK) and solidified on ice.
The plugs were placed in 2 ml microcentrifuge tubes (Fisher
Scientific, UK) and incubated overnight in 1 ml lysis buffer
with lysozyme (2 mg/ml) (Sigma-Aldrich, UK) in a 37°C
water bath with an agitation speed of 200 rpm. The following
day proteinase K (1 mg/ml) (Sigma-Aldrich, UK) was added
to each of the test tubes and incubated in a 55°C water bath
for 2 hours without agitation. The test tubes were inverted
occasionally (approximately every 30 minutes). To inactivate
proteinase K, the plugs were washed 4 times for 30 minutes in
1 ml t ris-EDTA buffer [10 m M tris-HCl (pH 8.0), 1 mM
EDTA (pH 8.0)] at 4°C.
Pseudomonas Aeruginosa
P. aeruginosa colonies were incubated overnight on nutrient
agar. Bacterial cells were suspended in 5 ml PET-IV buffer
(10 mM Tris-HCl (pH 7.5), 1 M NaCl). A spectrophotometer
(CE 1011, Cecil Instrument Ltd., UK) was set at a wavelength
of 600 nm and the bacterial cell density was adjusted to 1.5
using the PET-IV buffer. The bacterial suspension in PET-IV
buffer (2 ml) was dispensed in a sterilised centrifuge tube and
centrifuged at 9289 g for 5 minutes. The super natant was
removed carefully using a pipette and re-suspended in 2 ml of
PET-IV buffer, this procedure was repeated once more. A 2%
w/v low melting point agarose was prepared in water and
dissolved completely using a hot plate with constant stirring.
The prepared low melting point agarose was placed in a
heating block at 50°C.
The bacterial cell suspension in PET-IV (0.5 ml) was mixed
with 0.5 ml of the prepared 2% w/v low melting-point agarose
(approximately 50°C), transfer red to a plug mould and
solidified on ice. The plugs were incubated in 2.5 ml lysis
buffer (6 mM tris, 0.1 M EDTA, 1 M NaCl, 0.5% (w/v) Brij 58
(polyethylene glycol hexadecyl ether), 0.4% (w/v) sodium
deoxycholate, 0.5% (w/v) sodium lauryl sarcosine, pH was
adjusted at 6.4 using HCl) with lysozyme (1 mg/ml) for 24
hours at 37°C. The plugs were rinsed with sterilised distilled
water and incubated with 2.5 ml proteolysis buffer (0.5 M
EDTA (pH 8.2), 1% (w/v) sodium lauryl sarcosine) with
proteinase K (50 µg /ml) for overnight at 50°C. The plugs
were washed 4 times for 30 minutes with 2.5 ml of tris-EDTA
(TE) buffer [10 mM tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)].
DNA plugs were stored in 5 ml TE buffer at 4°C and used
within 5 weeks.2 6-30
         
B. cereus







Bacillus Cereus
The pulsed-field gel electrophoresis (PFGE) protocol for B.
cereus DNA sequencing was provided by Dr. Babetta L.
Marrone and Yulin Shou, Los Alamos National Laboratory,
Los Alamos, USA.24 ,25 B. cereus colonies were cultured
overnight on a solid nutrient media (CM0003, Oxoid, UK).
Single colony, collected from the overnight grown culture,
was added to 5 ml TSB and incubated for 14-16 hours in a
37°C water bath with an agitation speed of 200 rpm. The
following day, 5 µl of the overnight grown bacterial culture,
was added to 5 ml fresh TSB and incubated for a further 4
hours in a water bath with an agitation speed of 200 rpm. The
grown bacterial culture was centrifuged (Centrifuge 5804R
Eppendorf, Germany) at 3200 g and washed in 1 ml PET-IV
buffer (10 mM tris-HCl (pH 7.5) 1 M LiCl at) at 8228 g. The
iSoLation anD iDentiFiCation oF BaCteria 51

DNA extracted from B. cereus was digested using 20 units
(10000 units/ml) of SmaI restriction enzyme (Sigma-aldrich,
UK) in 200 µl 1 x concentrations SH buffer (Sigma-Aldrich,
UK) for 2 hours in a 25°C water bath with an agitation speed
of 200 rpm. Alternatively, DNA extracted from P. aeruginosa
was digested using 15 unit s (10000 units/ ml) of SpeI
restriction enzyme (Sigma-Aldrich, UK) in 100 µl of 1x
concentration SA buffer (Sigma-Aldrich, UK) overnight at
37°C.26-31

Electrophoresis was conducted using a PFGE system to
separate the enzyme-digested B. cereus and P. aeruginosa
DNA. The DNA band patterns obtained were compared with
the DNA band patterns of the control bacterial species (B.
cereus ATCC11778 and P. aeruginosa ATCC10145). A
lambda ladder (48.5 - 970 Kb, Bio-Rad, USA) was used as a
marker. A 1% w/v certified megabase agarose was prepared
in TBE buffer (0.5 x concentrations). Ethidium bromide (50
µg/ml) was added to the molten agarose. Digested plugs and
lambda ladder were loaded into the gel and sealed with 1%
w/v molten agarose. The agarose gel was then placed in a
clamped homogenous elect ric field (CHEF-DR II) system
(Bio-Rad, USA). The electrophoresis was carried out at 14°C
using 0.5 x concentration TBE buffer at 6 V/cm.
The running time for analysing P. aeruginosa DNA was 20
hours (block 1) with switch time ramped from 5 seconds
(initial switch time) to 45 seconds (final switch time) and for
a further 4 hours (block 2) with an initial switch time 45
seconds to a final switch time 90 seconds. The electrophoresis
for B. cereus DNA was conducted for 20 hours with an initial
switch time 2.2 seconds and a finial switch time 54.2 seconds.
The gel was visualised under UV (336 nm) using a Gel Doc
(Bio-Rad, USA) and photographed.27-30
Results And dIscussIon

Bacillus Cereus

        

        B. cereus     
B. cereus


B. subtilisB.
cereus         
   
B. cereus

B.
cereus
      B. cereus     
B. cereus
Pseudomonas Aeruginosa
P. aeruginosa
         P.
aeruginosa    P.
aeruginosa
       P. aeruginosa
  Pseudomonas     
P. aeruginosa

JALCA, VOL. 108, 2013
P. aeruginosaPseudomonas CN agar
media
B. cereus   


     
    


B. cereus
52 iSoLation anD iDentiFiCation oF BaCteria



The Biolog® System for Bacterial Identification



B. cereusP. aeruginosa
       
B. cereusP. aeruginosa

        
B. cereus B. thuringienisis
 B. cereus, B. thuringienisis  B.
anthracis 





  




P. aeruginosa
             
Bacillus     

        



Bacillus Cereus
B. cereus

      B. cereus
  
B. cereus
  
B. cereus

B.
cereus
B. cereus
    
B. cereus
     



JALCA, VOL. 108, 2013

B. cereus
B. cereus

 







   
   B. cereus
   B. cereus
   B. cereus
   B. cereus
   B. cereus
   B. cereus
   B. cereus

   


iSoLation anD iDentiFiCation oF BaCteria 53
JALCA, VOL. 108, 2013

B. cereusP. aeruginosa
B. cereus
P. aeruginosa
Isolated from
B. cereus P. aeruginosa
Biolog Identification Probability (%) Biolog Identification Probability (%)
 B. cereus/ thuringiensis   
    
 B. cereus/ thuringiensis   
 B. cereus/ thuringiensis  
 B. cereus/ thuringiensis  
    
 B. cereus/ thuringiensis  
   

    

B. cereus
B. cereus
 







   
   B. cereus
   B. cereus
   B. cereus
   -
   B. cereus
   B. cereus
   B. cereus
   -

  


54 iSoLation anD iDentiFiCation oF BaCteria
JALCA, VOL. 108, 2013

B. cereusP. aeruginosa
B. cereus
P. aeruginosa

 
   
 B. cereus/ thuringiensis   
 B. cereus/ thuringiensis   
 B. cereus/ thuringiensis  
 B. cereus/ thuringiensis  
 B. cereus/thuringiensis   
 B. cereus/ thuringiensis   
 B. cereus/ thuringiensis   
  

 B. cereus/ thuringiensis   
     
      
             
         
    

         
        
     

et alet al
   


    
    



  




              
     
              

         
       
    

   








               
B. cereusP. aeruginosa
     

B. cereusP. aeruginosa
     
B. cereus P. aeruginosa

            B. cereus   P.
aeruginosa            

iSoLation anD iDentiFiCation oF BaCteria 55
JALCA, VOL. 108, 2013
P. aeruginosa

P. aeruginosa     
P. aeruginosa


      Bacillus cereus 
B. cereus
B. cereus



Figure 6. Pulsed-field gel electrophoresis of P. aeruginosa DNA
(conventional leather making process). Lanes 1, 7 and 8: Lambda Ladder
(48.5- 970 kb), lane 2: P. aeruginosa ATCC10145 (control), lanes 3, 4,
5 and 6: DNA of P. aeruginosa isolated during the conventional pre-
soaking, soaking, deliming and bating processes respectively.
Figure 7. Pulsed-field gel electrophoresis of Bacillus cereus DNA (BAT
leather-making process). Lane 1: B. cereus ATCC 11778 (control); lanes
2, 3, 4, 5, 6, 7, 8 and 9: B. cereus DNA isolated during the conventional
pre-soaking, soaking, unhairing, hair from the unhairing effluent, re-
liming, deliming, bating and pickling processes respectively; lanes 10:
Lambda Ladder (48.5- 970 kb).
56 iSoLation anD iDentiFiCation oF BaCteria
JALCA, VOL. 108, 2013


      


   

 




  
  


                
      





 



B. cereus


 B. cereus
 
   
 
 
 
 
  
  
  
 

  
            

   





conclusIon

       


   
        

iSoLation anD iDentiFiCation oF BaCteria 57
JALCA, VOL. 108, 2013

P. aeruginosa


Processes Estimated number of recovered P. aeruginosa colony forming units/ ml
Conventional BAT Conventional BAT
Pre-soaking 107107105105
Soaking 107106-107105105
Unhairing/ liming 0 0 0 0
Re-liming 0 0 0 0
Deliming 0-1020-1040 0
Bating 0-1020-1040 0
Pickling 0 0 0 0-102
Chrome-tanning 0 0 0 0
Hair from the
unhairing eff luent 0 0-101- -
Acknowledgement

         




RefeRences
        
       Journal of
Society of Leather Technologists and Chemists

         
      
Journal of the Society of the Leather Technologists and
Chemists
    

JALCA
       

JALCA
 
JALCA 
        
      
JALCA
        
        
JALCA
 

JALCA
            
  
      JALCA 

  Clinical microbiology, an Introduction for
healthcare professionals   


         
      
  International Biodeterioration and
Biodegradation
Microbiology
concepts and application

58 iSoLation anD iDentiFiCation oF BaCteria
JALCA, VOL. 108, 2013
Microbiology

Aerobic plate count
at 30°C: spiral plate method (F11)  
     
      

    Preparation of
samples and dilutions (F2)   
     


    Enumeration of
Bacillus cereus and other Bacillus  (F15)
     
      

    Enumeration of
Pseudomonas aeruginosa by membrane filtration (W
6)
       
      

         Brock
biology of microorganisms
Medical
microbiology      

       Cowan and
Steel’s: Manual of the identification of medical bacteria

    Biolog, MicrologTM Ststem, Release 4.2
User Guide


    Appliedand
Environmental Microbiology

    Pharmacopeial Forum

        


     
 Journal of Clinical Microbiology

    
 Bacillus anthracis B. cereus  B.
thuringiensis     
Applied and Environmental Microbiology 

        
          
   Pseudomonas aeruginosa
Burkholderia cepacia
Staphylococcus aureus     
Journal of Cystic Fibrosis
     
    
 
Archives of Medical Research
       
Pseudomonas aeruginosa
      
International Journal of Antimicrobial Agents

  
  Pseudomonas aeruginosa   
Applied and Environmental
Microbiology
        
Pseudomonas aeruginosa
    
Microbial Pathogenesis



   Pseudomonas
aeruginosa Antimicrobial
Agents and Chemotherapy
    
Applications and systematics of Bacillus
and relatives

Journal of Clinical
Microbiology
      
    Escherichia coli  
      Journal of
Clinical Microbiology
        
         
Bacillus cereusPseudomonas aeruginosa
Staphylococcus aureus
    
JALCA
iSoLation anD iDentiFiCation oF BaCteria 59
JALCA, VOL. 108, 2013
AppendIx



Process Chemicals Amount
(% w/w)* Time pH Temperature
(°C)
Pre-soaking
Water
Truposept BA
(Trumpler, Germany)
300
0.2
60 mins 7.6-8.0 21.1-25.8
Main-soaking
Water
CorileneW385 (STAHL
Europe, The Netherlands)
Truposept BA
300
0.2
0.2
120 mins and
left overnight 7.6-8.1 21.5-25.6
Unhairing
Water
Sodium sulfide
Lime
200
3
2
20 -24 hrs 11.1-12.7 21.2-27.8
Washing Water 300 10 mins
Re-liming
Water
Lime
200
2
72 hrs 12.5-12.6 20.6-25.8
Washing
(three times) Water 300 10 mins
Deliming
Zero float
Ammonium chloride
Water
2
100
15 mins
30-45 mins
8.2-8.8 24.5-26.8
Bating Oropon ON 2 (TFL Germany) 0.1 30 mins 8.0-8.7 24.9-26.8
Washing (twice) Water 300 10 mins
Pickling
Water
Sodium chloride
Sulfuric acid
Formic acid
100
8
1.2
1.0
3-4 hrs and left
overnight 1.5-3.1 20.4-26.6
Tanning and
Basification
Chromium (III) sulphate
Feliderm®MGO
(Clariant, Switzerland)
8
0.4
90 mins
4 hrs
4.1-4.9
22.8-27.7

60 iSoLation anD iDentiFiCation oF BaCteria
JALCA, VOL. 108, 2013


  
   
 


   










  










  

   
 


   

   
 


   







   
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iSoLation anD iDentiFiCation oF BaCteria 61
... For air samples, each plate was exposed to air for 60 min; aliquots from water, soil and feeds were seeded onto the media plates while hand swabs were inoculated onto the plates. After incubation for 24 h at 30 o C, the bacterial species were identified based on their colony appearances (Lama et al., 2013). The colonies on each plate were counted with magnifying glass. ...
Bacterial Profile of Livestock Farms in South-East Nigeria
Article
Full-text available
  • Sep 2022
The unregulated practice of livestock production has endangered the public health sector through the multiplication and spread of bacterial pathogens. This study investigated the bacterial profiles of livestock farms in Aba, Umuahia, Okigwe and Mbaise in the Southeastern part of Nigeria. Air was sampled with passive sedimentation technique; water samples were collected randomly from the farm water sources while hand swabs from the farmers and feeds were collected with sterile swab sticks and containers respectively. Total heterotrophic bacterial count (THBC) was analyzed by pour plate method; total coliform count (TCC) was determined by membrane filter technique while total potential pathogenic bacterial count (TPPBC) was examined by growing the samples in some selective agar media. Of the four cities studied, Aba had the highest THBC (28.43±0.3×105, 26.70±0.7×105, 26.26±0.5×105 CFU/ml), TPPBC (17.47±0.5×105 CFU/ml and 20.02±0.5×105 CFU/ml) and TCC (24.06±0.4×105, 17.93±0.6×105 and 22.36±0.4×105 CFU/ml) for pig, cow and poultry farms respectively while Mbaise had the least value. A total of thirteen (13) bacterial species were isolated in the study but, only Escherichia coli, Staphylococcus aureus, Klebsiella sp., Salmonella sp., Proteus mirabilis and Bacillus subtilis were commonly distributed in the four cities. Bacillus subtilis, Salmonella sp. and Staphylococcus aureus were isolated more in Okigwe, Aba and Umuahia respectively than Mbaise. Salmonella sp. (60.00%) had the highest occurrence followed by Staphylococcus aureus (55.33%) while Proteus mirabilis (4.50%) had the lowest occurrence. High bacterial loads were obtained in the study especially in Aba. Livestock farmers should consider proper hygienic measures in order to limit the spread of pathogenic bacteria among surrounding communities.
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... Even so, many weaknesses have been noticed in the SCcuring (Bailey, 2003). For example, the destructive role of bacteria (Cadirci et al., 2010;Berber and Birbir, 2010;Birbir, 2011b, 2012;Lama et al., 2012Lama et al., , 2013Ulusoy and Birbir, 2015), especially halophilic bacteria (Bailey and Birbir, 1996;Shede et al., 2008;Berber et al., 2010c;Bilgi et al., 2015;Akpolat et al., 2015;Caglayan et al., 2015) has become more realistically appraised in the hide curing and storage process. Halophilic bacteria contain special lipases and proteases that can potentially digest substances in SC-cured hides, and it can result in the "red heat", one of the most common disadvantages of raw hides and skins (Akpolat et al., 2015). ...
Recent Progress in Cleaner Preservation of Hides and Skins
Article
The hides and skins are flayed from animals and further processed into leathers. As the main constituent of hides and skins is protein, these materials are highly susceptible to bacterial action. Therefore, adequate preservation of hides and skins is crucial in slaughterhouses and tanneries. Preservation using sodium chloride (SC) remains the most popular curing technique worldwide due to its ease, cost-effectiveness and the quality of the finished leather produced. The use of SC (approximately 40-50% on raw hides/skins weight) enhances the pollution load of tannery effluent, however, which becomes highly contaminated with increased total dissolved solids (TDS) and chlorides. To overcome this hurdle, researchers are constantly searching for alternative preservation techniques which are either totally void of SC or use only a small amount of SC. Based on the literatures published mainly in the past decade, this review systematically and comprehensively summarizes current status and development trend about the cleaner preservation methods used in curing and soaking processes in leather industry. The discussed sodium chloride less curing methods contain SC + EDTA, SC + silica gel, SC + sodium meta-bisulphite and SC + boric acid, while the illustrated alternatives used in sodium chloride free curing methods include inorganic preservatives (potassium chloride, sodium sulphate, silicate and ozone), organic preservatives (natural and synthetic preservatives) and other chemical antiseptics. Emphasis is put on natural preservatives (paste plant formulations, essential oils, chlorophyll and bacteriocins), polyethylene glycol, ozone and physical preservative methods containing electric current, chilling and vacuum. Furthermore, several proposals are addressed for the development of eco-friendly and efficient preservation methods for hides and skins.
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INVESTIGATION OF BACTERIAL CONTAMINANTS IN FREEZERS KEEPING FROZEN FOOD IN LOCAL MARKETS
Article
Full-text available
  • May 2017
This study aimed to evaluate the occurrence of microbial contamination in food keeping freezers in some local markets in Baghdad city/ Iraq, as well as the contamination of the hands of workers in markets, and the possibility of contamination caused by the transport of food. 30 samples of snow ice found in food keeping freezers in local markets was randomly collected, and 30 swabs from workers hands were taken from the same markets at the same time. Microbiological examination of ice samples was conducted as well as the hands of workers’swabs, and the bacteria were isolated and diagnosed through microbiological and biochemical tests followed. Microbial test results showed some isolates of bacteria in ice samples obtained from food keeping freezers, the highest number of isolates belonging to Pseudomonas aeruginosa 12 isolates, followed by Klebsiella spp bacteria reaching 7 isolates, then Staphylococcus aurous recorded 4 isolates, while the number of isolates of each bacteria belonging to Staphylococcus epidermidis, Salmonella spp, Streptococcus fecalis and Escherichia coli is equal: 2 isolates,and one isolate from Escherichia coli O157:H7 . In the swabs of workers’ hands the numbers for each of the isolates of bacteria Klebsiella spp., E.coli, Staphylococcus aurous, Streptococcus fecalis, Staphylococcus epidermidis and Escherichia coli O157:H7 recorded 10, 10, 7, 5, 4 and 2 respectively, and Salmonella spp. pseudomonas aeruginosa bacteria not found in the swabs of workers’ hands
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The effect of conservation defects on the suede quality of double-face
Article
Full-text available
In this research, the effects of microorganisms on suede quality of double-face leather were examined. The bacterial numbers on salted sheepskin samples were found as 1.8×106-4.3×107 cfu/g on solid medium and 1.9×106-4.7×107 cfu/g on halophile medium containing 10-30% NaCl. However it was found that salted sheepskins contained 4.4×101-2.0×104 cfu/g of fungi. While 53-74% of halophilic bacteria and 39-58% of fungi showed proteolytic activity, 47-62% of halophilic bacteria and 35-54% of fungi showed lipolytic activity. It was observed that defective light stained areas on suede surface of finished double-face leathers were caused by microorganisms on salted sheepskins and that these defects correspond to 35% of the total double-face leathers amount. Consequently, it was decided that the proteolytic and lipolytic activities of microorganisms on the flesh side of the sheepskins decreased the suede quality and caused economical losses.
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Examination of bacterial populations in salt, salted hides, soaked hides and SOAK liquors
Article
Full-text available
This study was performed to evaluate the efficiency of salt curing and soaking methods containing antibacterial agents. The bacteria adversely affecting hide quality because of hide preservation method and soaking process were examined in salt (n=40), salted hide (n=36), soaked hide (n=34) and soak liquor (n=19) samples obtained from different tanneries in Leather Organized Tannery Region, Tuzla-i̇stanbul, Turkiye. Most of the hides examined in this study (83%) were imported from abroad. The pH, moisture content %, ash content % and salt saturation % of the salted hides were determined. In the salted hides, the pH values of all samples (pH 6-9) and moisture content (49-66%) in 28% of the samples were found to be appropriate for bacterial growth. It was determined that ash content in 25% (10-14%) of the salted hides and salt saturation in 22% (65-84%) of the salted hides were suitable for the growth of mesophilic bacteria. Despite the salt-curing of hides, proteolytic and lipolytic mesophilic bacteria were isolated from the hides in high numbers. Proteolytic and lipolytic extremely halophilic bacteria were observed in the most samples of salt and salted hides. These bacterial counts were 10 2-10 4 c.f.u./g in the salt samples whereas they were 10 2-10 6 c.f.u./g in the salted hides. Although proteolytic and lipolytic mesophilic bacteria were 10 5-10 8 c.f.u./g in 97% of the soaked hide samples, they were 10 5-10 6 c.f.u./ml only in 42% of the soak liquors. In conclusion, it was determined that this hide preservation method was not adequate to inactivate bacterial activity. Hence, bacterial activity was high in the salted and soaked hides and also the concentration of antibacterial agents used in the soaking process was not sufficient. It is believed that eliminating of these problems will provide to increase hide quality.
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Measurement of bacterial populations in typical tannery soak solutions by traditional and new approaches
Conference Paper
It has been well documented that bacteria will readily proliferate during the typical soaking process if left unchecked. Tanners are rightfully concerned about the extent of biological activity in their soaking process and the impact this can have on the grain integrity and strength of the resulting leather. However, inquiries such as what level of microbial activity is a cause for alarm or is there a threshold activity limit below which it can be considered acceptable abound. A project was undertaken to survey several of the largest beamhouses in the USA to determine the level of microbial activity at each location as well as differences in nature of the raw material utilized and the type of control programs in place (if any). Data was collected utilizing traditional and new techniques for measurement. The results are analyzed and conclusions are drawn regarding the extent of biological activity present in typical operating conditions in the different tanneries studied.
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Applications and Systematics of Bacillus and Relatives
Book
  • Jan 2008
Inspired by the pace of change in the taxonomy of the aerobic endospore-forming bacteria, the "Bacillus 2000" symposium on which this book is based was held in Bruges, Belgium, in August 2000, and was supported by the Federation of European Microbiological Societies, the Belgian Society for Microbiology, and several commercial sponsors. Bringing taxonomists interested in Bacillus and its relatives together with people who work with these organisms in medicine, agriculture, and industry, allowed those attending to appreciate the overlaps and interactions of their areas of expertise, in the absence of any comprehensive treatment of the current systematics of the group. The meeting was a great success, and has resulted in the production of these proceedings, Applications and Systematics of Bacillus and Relatives, providing an up-to-date and comprehensive treatise on the classification, identification and applications of the aerobic endospore-forming bacteria; it is an essential reference for all microbiologists interested in these organisms. Valuable reference work for all those interested in the systematics of Bacillus and its relatives. Produced in response to the successful Bacillus 2000 meeting in Bruges and was supported by the Federation of European Microbiological Societies, the Belgian Society for Microbiology, and several commercial sponsors. Of use to those working in fields as diverse as medicine, agriculture, food and industry. Comprehensive and up-to-date analysis of the systematics of these organisms. Includes the application of sophisticated chemotaxonomic and genetic characterization methods.
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Isolation and identification of bacteria adversely affecting hide and leather quality
Article
  • Sep 1996
Two hundred and twenty five hide and chrome tanned leather samples were examined at different stages of processing to characterize the populations of organisms inhabiting them. Bacillus subtilis, Bacillus cereus, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus sphaericus, Micrococcus luteus, Staphylococcus aureus, Micrococcus rubens, Kurthia variabilis, Pseudomonas aeruginosa, Staphylococcus epidermidis, Bacillus brevis, Micrococcus candidus, Micrococcus roseus, Bacillus firmus and Bacillus laterosporus were isolated and identified in different stages of tannery processing. Bacillus brevis, Bacillus cereus, Bacillus firmus, Bacillus laterosporus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus sphaericus, Bacillus subtilis, Micrococcus luteus, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis were positive for gelatin hydrolysis. Fresh and salted hide samples were also evaluated chemically and histologically. Some of the salted hide samples showed histological changes resulting from poor curing techniques and high moisture content. It has been noted that generally Bacillus species caused deterioration of hides. It is concluded that the hide damage is caused by inadequate curing, bacterial action and autolysis.
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Pulsed-Field Gel Electrophoresis (PFGE) Technique and its use in Molecular Biology
Article
  • Jan 2001
In recent years, the use of pulsed-field gel electrophoresis (PFGE) in the molecular biology area has been subject to much research. PFGE is a powerful tool for characterizing various strains at the DNA level, obtaining relevant information on genome size and constructing the physical and genetic map of the chromosome of bacteria that are poorly understood at the genetic level as well as in separating chromosomes in microorganisms, and in the long-range mapping of mammalian genes. PFGE also has advantage of examining the elongated and oriented configuration of large DNA molecules in agarose gels at finite field strengths. In this review, the use of PFGE in molecular biology, the general characteristics of PFGE, different types of PFGE and factors affecting PFGE are introduced. Özet: Son y›llarda, moleküler biyoloji alan›nda pulsed-field jel elektroforez (PFGE)'in kullan›m› birçok araflt›rmaya konu olmufltur. PFGE, DNA düzeyinde çeflitli strainlerin karakterize edilmesinde, genom büyüklükleri hakk›nda bilgi elde etmede, genetik düzeyde anlafl›lamam›fl bakteri kromozomlar›n›n fiziki ve genetik haritalar›n›n oluflturulmas›nda,mikroorganizmalar›n kromozomlar›n›n ayr›lmas›nda, ve memeli genlerinin büyük çaptaki haritalanmas›nda kullan›lan etkili bir metottur. PFGE; agaroz jel içerisindeki büyük DNA moleküllerinin uygun bir alanda deiflik flekillerde hareket etmesini salayan bir avantaja da sahiptir. Bu derlemede, PFGE'in moleküler biyoloji alan›nda kullan›m›, PFGE'in genel özellikleri, çeflitli tipleri ve PFGE'i etkileyen faktörler sunulmufltur.
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Application of molecular techniques to identify bacteria isolated from the leather industry
Article
DNA-based methods are known to be accurate, precise and rapid in identifying bacteria but there is little information about the use of these techniques in the leather industry. A study was undertaken to investigate and characterize the bacterial contaminants of fresh hides using 16S ribosomal RNA sequencing techniques. The proteolytic activities of these isolates were also studied using different substrates. Organisms belonging to 16 different genera of bacteria were isolated, and many of the species found have not been previously reported for this environment. Some of the species of bacteria being reported for the first time in the tanning environment included Janibacter terrae, Acidovorax sp., Dietzia maris, Arthrobacter pr tophormiae, Comamonas sp., Brevibacterium lutescens, Jeotgalicoccus psychrophilus, Nocardiopsis sp., Delftia acidovorans, Pseudomonas cannabina, Pseudomonas fulgida and certain species of Aeromonas and Acinetobacter. Most species of bacteria found in the incoming process water were non-proteolytic. Analysis of fresh hides revealed a large number of both proteolytic and non-proteolytic bacteria. The proteolytic bacteria included several species of Aeromonas, Proteus vulgaris, and Shewanella putrefaciens. Pseudomonas sp. were common but not all of them showed proteolytic activity. Knowledge about the identities and proteolytic activities of these organisms that occur in this environment would provide a better way to design programs to control theirundesirable activities.
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