# Presentation: 879958

## Concentration of pathogens and indicators from large sample volumes using continuous flow centrifugation

- Fu-Chih Hsu, R. Wong, J. Larkin, and D.A. Battigelli

## Study objectives

- Validate CFC for EPA Method 1623
- Mean recovery and precision
- Procedures evaluation and improvement
- Matrix impacts
- Comparison to other filtration based methods

**Notes:**
This is very interesting study done recently.  TC indicates total coliforms, FC fecal coliforms and En indicated enterococci.  If any of them show in one site, it indicate the concentration of that indicator is over the limit. In sites 1 and 8, concentrations of all three bacterial indicators are over the limit, but no adenovirus is detected.  Sites 4, and 5 meet the bacterial indicator standards, but are positive for adenoviruses. Somatic coliphages were detected in all sites.  It is clear that F+ is a better indicator for predicate adenoviruses because there is about 75% agreement between F+ and adenoviruses in this study.

## Study objectives

- Simultaneous concentration of protozoa, bacteria and viruses
*Crypto*/*Giardia*/bacteria should be concentrated inside the CFC bowl
- Viruses may be captured after CFC using positive charged filters
- CFC as prefiltration
- Larger water sample tested

**Notes:**
This is very interesting study done recently.  TC indicates total coliforms, FC fecal coliforms and En indicated enterococci.  If any of them show in one site, it indicate the concentration of that indicator is over the limit. In sites 1 and 8, concentrations of all three bacterial indicators are over the limit, but no adenovirus is detected.  Sites 4, and 5 meet the bacterial indicator standards, but are positive for adenoviruses. Somatic coliphages were detected in all sites.  It is clear that F+ is a better indicator for predicate adenoviruses because there is about 75% agreement between F+ and adenoviruses in this study.

## Why concentrate multiple microorganisms

- Grab samples vs. composite samples
- Decrease sample variations
- A better tool to collect samples for study relationships between indicators and pathogens
- Emerging response for biological attack
- Investigation of waterborne outbreaks

**Notes:**
This is very interesting study done recently.  TC indicates total coliforms, FC fecal coliforms and En indicated enterococci.  If any of them show in one site, it indicate the concentration of that indicator is over the limit. In sites 1 and 8, concentrations of all three bacterial indicators are over the limit, but no adenovirus is detected.  Sites 4, and 5 meet the bacterial indicator standards, but are positive for adenoviruses. Somatic coliphages were detected in all sites.  It is clear that F+ is a better indicator for predicate adenoviruses because there is about 75% agreement between F+ and adenoviruses in this study.

## Introduction

- Concentration Methods
- Dialysis
- Precipitation
- Filtration
- Batch centrifugation
- Continuous Flow Centrifugation (CFC)

## Filtration

- High flow rate
- Turbidity
- Porosity
- Cost

## CFC

- Flow rate: typically 100 mL to 700 mL per min, but can be higher
- Turbidity
- Multiple microorganism concentrations
- Cost

## Principle of CFC

*Principle of CFC*

**CFC Bowl**

**Inlet**

**Outlet**

## Principle of CFC

*Principle of CFC*

**Inlet**

**Outlet**

## Principle of CFC

*Principle of CFC*

**Inlet**

**Outlet**

## Principle of CFC

*Principle of CFC*

**Outlet**

**Inlet**

## Field-deployable version of CFC 

**CFC bowl**
**10,000 rpm**

**Pumps**
**Adjustable flow rate**

**Control board**

## Procedure

*Cryptosporidium*, *Giardia*,
- Bacteria, Virus

**Water**

- Pump

- Pump

- Virus capture filter

- Waste

- Waste

**CFC**

## Procedure-Virus absorption

- 		 * * Virus (coliphage MS2)
- Positively charged filter
- 1MDS, Nanoceram
- No requirement for conditioning of water
- Relatively large pore size
- Cost-effective

**Notes:**
This is very interesting study done recently.  TC indicates total coliforms, FC fecal coliforms and En indicated enterococci.  If any of them show in one site, it indicate the concentration of that indicator is over the limit. In sites 1 and 8, concentrations of all three bacterial indicators are over the limit, but no adenovirus is detected.  Sites 4, and 5 meet the bacterial indicator standards, but are positive for adenoviruses. Somatic coliphages were detected in all sites.  It is clear that F+ is a better indicator for predicate adenoviruses because there is about 75% agreement between F+ and adenoviruses in this study.

## 1 liter seeding studies usingelectropositive filters

- 		 * *

- _Water Sample_ 			_% Recovery _
- River water 				47
- Ground water 				50
- Sea water	 				69-75

## Procedure-Elution

*Cryptosporidium*, *Giardia*, Bacteria
- 0.1 X PBS/0.01% Tween 80
- Shaking 5 min x 3
- Total final volume: 250 mL
- 	* *

**Notes:**
This is very interesting study done recently.  TC indicates total coliforms, FC fecal coliforms and En indicated enterococci.  If any of them show in one site, it indicate the concentration of that indicator is over the limit. In sites 1 and 8, concentrations of all three bacterial indicators are over the limit, but no adenovirus is detected.  Sites 4, and 5 meet the bacterial indicator standards, but are positive for adenoviruses. Somatic coliphages were detected in all sites.  It is clear that F+ is a better indicator for predicate adenoviruses because there is about 75% agreement between F+ and adenoviruses in this study.

## Procedure-Elution

- 		Virus (coliphage MS2)
- 3% Beef Extract/0.25M glycine/0.1% Tween80
- Add 2 mL elution buffer to the filter for 5 min, followed by additional 2 and 1 mL
- Final volume: 5 mL.

**Notes:**
This is very interesting study done recently.  TC indicates total coliforms, FC fecal coliforms and En indicated enterococci.  If any of them show in one site, it indicate the concentration of that indicator is over the limit. In sites 1 and 8, concentrations of all three bacterial indicators are over the limit, but no adenovirus is detected.  Sites 4, and 5 meet the bacterial indicator standards, but are positive for adenoviruses. Somatic coliphages were detected in all sites.  It is clear that F+ is a better indicator for predicate adenoviruses because there is about 75% agreement between F+ and adenoviruses in this study.

## Detection

*Cryrptosporidium* and *Giardia*
- Centrifuge at 1500 x g for 15 min
- EPA Method 1623
- Total coliforms and *E. coli*
- mEndo and mTEC agar
- Virus: MS2 coliphage
- Double agar layer and single agar layer

## Cryptosporidium and Giardia Results

- _______________________________________________________
- _Sample type_	   _Turbidity__	_	_*Crypto *__(Recovery/RSD)_	 _*Giardia *__(Recovery/RSD)_
- IPR	NA		36 / 24 %		             37 / 24 %
- MS	2.6		69 / 13 %		             82 / 3   %
- PT	2.7	                 72 / 6 %		             56 / 28 %
- _____________________________________________________
- Spike conc:
- 	IPR and PT: 98-99 oocysts/csys per 15 L
- 	PT : 75-101 oocysts/cysts per 20 L
-  Flow rate: 700 mL per min

## E. Coli Results 

- Mean recovery from 2.5 liters of tap water_ _
- ___________________________________ _
- _Flow rate_	_Seeding (cfu)_	_Recovery (%)__ _
- 700 ml/min	     113 ± 6		     31 ± 3
- 100 ml/min	       96 ± 12		     87 ± 4
- ________________________________________

## Crypto/Giardia/virus Results

- ____________________________________________
- _Microorganism_		_Spiking*_		_Recovery__	__(%)_
*Cryptosporidium*		     149/10 L   		54 ± 5
*Giardia*			   152/10 L 		31 ± 2
- F+Coliphage **		 NA		46 ±14
- __________________________________________
- *: River water matrix with 700 mL per min flow rate
- **: Field male-specific coliphages; 47 mm positive charged filter per 2 liter

## Total coliform/E. coli/virus Results

- ____________________________________________
- _Microorganism_		_Spiking*_		_Recovery__	__(%)_
*Total coliform**	*		     225/ L   		76
*E. coli*			     13/ L 		 81
- Coliphage MS2**		     10/ L		 50
- __________________________________________
- *: ground water filtered by 100 mL per min
- **: 47 mm positive charged filter was used for every 2
-      liters

## Conclusions

- CFC is an excellent concentration method for Method 1623
- EPA approval
- Efficient recovery of microorganisms tested
- No filter clogging problem

##  Conclusion

- It is possible to concentrate multiple microorganisms using CFC
*Crypto*/*Giardia*/bacteria can be concentrated inside the CFC bowl
- Viruses can be captured and eluted by the positively charged filters after CFC

##  Conclusion

- Nanoceram filter
- High capacity: a 47 mm filter can filter more than 1 liter surface water and >2 liter of ground water
- A versatile filter: river, tap, ground, and sea water
- Inexpensive